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https://kar.kent.ac.uk/96256/1/Kallitsounaki_et_al-2022-Archives_of_Sexual_Behavior.pdf
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Implicit and Explicit Gender-Related Cognition, Gender Dysphoria, Autistic-Like Traits, and Mentalizing: Differences Between Autistic and Non-Autistic Cisgender and Transgender Adults
|
Archives of sexual behavior
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If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version. Author Accepted Manuscripts If this document is identified as the Author Accepted Manuscript it is the version after peer review but before type
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https://doi.org/10.1007/s10508-022-02386-5 The version of record is available from
https://doi.org/10.1007/s10508-022-02386-5 Kent Academic Repository Kent Academic Repository Kallitsounaki, Aimilia and Williams, David M. (2022) Implicit and Explicit
Gender Related Cognition, Gender Dysphoria, Autistic Like Traits, and
Mentalizing: Differences Between Autistic and Non Autistic Cisgender and
Transgender Adults. Archives of Sexual Behavior . ISSN 0004-0002. Downloaded from
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in KAR. If you believe that your, or a third party's rights have been compromised through this document please see
our Take Down policy (available from https://www.kent.ac.uk/guides/kar-the-kent-academic-repository#policies). Archives of Sexual Behavior
https://doi.org/10.1007/s10508-022-02386-5 ORIGINAL PAPER Abstract Evidence indicates a link between autism spectrum disorder (ASD) and gender diversity, yet this intersection remains
insufficiently understood. Here, we investigated whether (1) ASD affects gender-related cognition (i.e., mental processes of
perceiving and interpreting one’s own gender self-concept), (2) autistic people have increased gender dysphoria and recall
limited gender-typed behavior from childhood, and (3) transgender individuals have increased ASD-like traits and difficulties
in mentalizing. A total of 106 non-autistic cisgender (51 birth-assigned female), 107 autistic cisgender (57 birth-assigned
female), 78 non-autistic transgender (41 birth-assigned female), and 56 autistic transgender adults (27 birth-assigned female)
participated in the study. The mean age of participants was 31.01 years (range = 18 to 70). Using an explicit as well as an
implicit measure, for the first time, we found that ASD affected gender-related cognition only in autistic cisgender people. Sex differences were also observed in this group. Whereas autistic cisgender birth-assigned males showed a stronger implicit
gender-group identification than non-autistic cisgender birth-assigned males, autistic cisgender birth-assigned females showed
a weaker gender-group identification than non-autistic cisgender birth-assigned females. Furthermore, autistic cisgender people
reported significantly more gender dysphoric feelings and recalled significantly less gender-typed behavior from childhood
than non-autistic cisgender individuals. No difference was observed between non-autistic and autistic transgender people. We
also found that relative to non-autistic cisgender individuals, both non-autistic transgender and autistic transgender people
reported significantly more ASD-like traits. However, mentalizing difficulties were observed only in the latter group. This
research enhances our understanding of the link between ASD and gender diversity. Keywords Gender cognition · Autism spectrum disorder · Transgender · Mentalizing · Autistic-like t * Aimilia Kallitsounaki
A.Kallitsounaki-836@kent.ac.uk Implicit and Explicit Gender‑Related Cognition, Gender Dysphoria,
Autistic‑Like Traits, and Mentalizing: Differences Between Autistic
and Non‑Autistic Cisgender and Transgender Adults Aimilia Kallitsounaki1 · David M. Williams1 Received: 24 August 2021 / Revised: 10 June 2022 / Accepted: 18 July 2022
© The Author(s) 2022 Received: 24 August 2021 / Revised: 10 June 2022 / Accepted: 18 July 2022
© The Author(s) 2022 1
Division of Human & Social Sciences, School
of Psychology, Keynes College, University of Kent,
Canterbury CT2 7NP, UK Research Question 1: Does Autism Spectrum
Disorder Affect Implicit and Explicit Gender‑Related
Cognition? Adopting an individual differences approach, however, Kallit-
sounaki and Williams (2020a) examined how the strength of
gender self-concept varies according to the number of ASD-
like traits manifested by people from the general population,
using not only an explicit measure, but also an Implicit Asso-
ciation Test (IAT; Greenwald & Farnham, 2000). It is well-
established that ASD-like traits are continuously distributed
in the general population and qualitatively similar to the core
diagnostic characteristics of ASD (e.g., Constantino & Todd,
2003, 2005; Ronald et al., 2006; though see Mottron, 2021 for
an alternative perspective). Hypothesis 1b Within each group (autistic cisgender/autistic
transgender/non-autistic cisgender/non-autistic transgender)
scores on the explicit and the implicit measures of gender
self-concept would align with experienced gender, rather
than birth-assigned sex (i.e., individuals who identify as
females would show an explicit and implicit female self-
concept, whereas people who identify as males would show
an explicit and implicit male self-concept). Hypothesis 1c We expected autistic participants who identify
either as cisgender or transgender to show a significantly
weaker explicit and implicit gender self-concept than non-
autistic people. Following this approach, Kallitsounaki and Williams
(2020a) found that people from the general population with
high ASD-like traits had a weaker inclination to ascribe to
themselves explicitly, or identify implicitly with, feminine
and masculine gender stereotypical personality traits than
people with low ASD-like traits. An exploratory analysis,
however, showed a selective influence of ASD-like traits on
the implicit identification with gender stereotypical traits
among birth-assigned females only. Research Question 1: Does Autism Spectrum
Disorder Affect Implicit and Explicit Gender‑Related
Cognition? Little is known about the mental processes involved in the
formation and consolidation of gender self-concept (or gender
identity) in ASD. The sparse research conducted on this topic
has shown that autistic adults show a reduced propensity to
explicitly identify with personality traits and gender roles that
are stereotypically attributed to males (Bejerot & Eriksson,
2014; Stauder et al., 2011). It has also been found that autistic
people feel less positively and identify less strongly with the
gender group that matches their experienced gender than do
non-autistic individuals (Cooper et al., 2018). Yet, a note of
caution should be added here, as these findings are from self-
report measures only. Self-report measures rely on people’s
ability to represent and report their own feelings, emotions,
traits, and thoughts. Given that aspects of self-awareness
are considered to be divergent in ASD (e.g., Carruthers,
2009; Gopnik, 1993; Williams, 2010), it is unclear whether
these findings reflect a different implicit self-experience of
gender (and hence, accurate self-reporting) or difficulties in
the explicit representation and/or reporting of this concept. To address this issue, along with explicit measures of gender
self-concept, researchers could also use implicit measures
(Wood & Eagly, 2009). Hypothesis 1a The number of self-reported ASD-like traits
of non-autistic cisgender people would be negatively and sig-
nificantly associated with the strength of gender self-concept
(more ASD-like traits = weaker explicit and implicit gender-
group identification). Next, we attempted to extend the original findings further
by examining gender-group identification in autistic people. Autistic people’s sense of belonging to one gender group over
another was examined in cisgender and transgender people,
separately, for the first time. This enabled us to understand
how exactly ASD might affect gender-related cognition. Olson et al. (2015) were the first researchers to use an IAT
to examine gender-related cognition in the transgender
population. They found that transgender children implicitly
perceived themselves in keeping with their experienced
gender. To our knowledge, no study has employed an IAT in
transgender adults. On the basis of previous findings (e.g.,
Cooper et al., 2018; Kallitsounaki & Williams, 2020a; Olson
et al., 2015), we made the following hypotheses: To our knowledge, no study has used an implicit meas-
ure to investigate gender-related cognition in autistic people. Introduction is an umbrella term used to describe a wide range of aspects
of one’s experienced gender that do not correspond to one’s
birth-assigned sex (Corbett et al., 2022). Among others, the
term gender diversity includes people who identify with a
gender other than their birth-assigned sex (i.e., transgender;
Butler, 2020), including those who feel significant levels of
distress about a mismatch between their experienced gender
and their birth-assigned sex (i.e., gender dysphoria; Ameri-
can Psychiatric Association, 2013). In recent years, clinicians and researchers have provided
increasing evidence for a link between autism spectrum dis-
order (ASD) and gender diversity (e.g., Strang et al., 2018a;
for a review and meta-analysis, see Kallitsounaki & Williams,
2022). ASD is a neurodevelopmental condition, diagnosed on
the basis of behavioral difficulties with social-communica-
tion and restricted, repetitive pattern of interests and behavior
(American Psychiatric Association, 2013). Gender diversity Research has provided strong evidence that the co-occur-
rence of ASD and gender diversity is accompanied by an
increase in internalizing conditions (e.g., anxiety and depres-
sion), self-harm, and suicidality (George & Stokes, 2018a;
Mahfouda et al., 2019; Strang et al., 2021; Strauss et al.,
2021). This highlights the need for specialized and tailored
support and care for this group. To achieve this, it is important
to gain a holistic understanding of the intersection between (0121 Archives of Sexual Behavior The first aim of this study was to replicate these findings
conceptually and extend them further. Specifically, in this
study we examined gender-group identification using an
explicit self-report measure and an IAT (Greenwald et al.,
2002). Gender-group identification implicates people’s sense
of belonging to one gender group as opposed to another and is
considered as one of the two traditions of research on gender
self-concept (Wood & Eagly, 2015). Based on Kallitsounaki
and Williams’ findings, we made the following hypothesis: ASD and gender diversity. In the current article instead of
focusing on a single component of the link between ASD and
gender diversity, we examined three core aspects of it in the
same sample, for the first time. 3 Research Question 3: Do Transgender People Have
Increased Autistic‑Like Traits and Mentalizing
Difficulties? Using the Gender Iden-
tity/Gender Dysphoria Questionnaire (GIDYQ; Deogra-
cias et al., 2007), George and Stokes found that autistic
people reported significantly more gender dysphoric feel-
ings than non-autistic individuals. In George and Stokes’
study, however, the percentage of participants who did
not identify as cisgender was 3 times higher in the autis-
tic group than in the non-autistic group. The inclusion of
these participants in the analysis could have artificially
inflated the score of the autism group creating a signifi-
cant difference in the number of gender dysphoric feel-
ings between autistic and non-autistic people. To begin to
answer whether autistic cisgender adults have increased
gender dysphoric feelings, we examined gender dysphoric
feelings among autistic cisgender and autistic transgender
people, separately. We also examined, for the first time,
whether these groups recall limited gender-typed behavior
from childhood. On the basis of previous findings (e.g.,
Bejerot & Eriksson, 2014; Hisle-Gorman et al., 2019;
Pecora et al., 2020; Pohl et al., 2014; Walsh et al., 2018),
we made the following hypotheses: The third aim of the current study was to examine whether
transgender people (autistic and non-autistic) have increased
ASD-like traits and difficulties in mentalizing. Previous
studies have suggested there may be a selective difference
in ASD-like traits between transgender and control birth-
assigned females only (i.e., Jones et al., 2012; Kung, 2020;
Murphy et al., 2020; Vermatt et al., 2018). Similar results
were also found by Nobili et al. (2018). We should note,
however, that in this study cisgender participants’ score on
the Autism-Spectrum Quotient (AQ-28) was higher than
the score of people from the general population (Hoekstra
et al., 2011). This makes the interpretation of Nobili et al.’s
(2018) findings difficult. Lastly, in Pasterski et al.’s (2014)
study transgender birth-assigned females, diagnosed with
gender dysphoria/gender identity disorder, scored higher
on the AQ-50 than non-autistic birth-assigned females,
but the difference was small and nonsignificant. In contrast
to previous findings, Stagg and Vincent (2019) found that
transgender individuals scored significantly higher on the
AQ-50 than cisgender people, regardless of participant birth-
assigned sex. Likewise, using the Social Responsiveness
Scale for Adults (SRS), Heylens et al. (2018) found that birth-
assigned males diagnosed with gender dysphoria reported
significantly more ASD-like traits than a norm group of birth-
assigned males and birth-assigned females diagnosed with
gender dysphoria reported significantly more ASD-like traits
than a norm group of birth-assigned females. Lastly, Warrier
et al. Research Question 2: Do Autistic People Have
Increased Gender Dysphoric Feelings and Recall
Limited Gender‑Typed Behavior from Childhood? The second aim of this study was to examine whether
autistic people have increased gender dysphoric feelings
and whether they recall limited gender-typed behavior
from their childhood years. Recent evidence indicates a 3 3 Archives of Sexual Behavior Research Question 3: Do Transgender People Have
Increased Autistic‑Like Traits and Mentalizing
Difficulties? small association between ASD traits and gender vari-
ance in children from the general population (Munoz
Murakami et al., 2022), as well as an increased rate of
gender diversity among autistic children (Corbett et al.,
2022). Likewise, research among adults has shown that
autistic people report a more diverse range of gender iden-
tities than non-autistic individuals (Bejerot & Eriksson,
2014; George & Stokes, 2018b) and that they are more
likely to be planning or have transitioned (Cooper et al.,
2018). To date, only George and Stokes (2018b) have
employed a validated measure to examine gender dys-
phoric feelings in this population. Using the Gender Iden-
tity/Gender Dysphoria Questionnaire (GIDYQ; Deogra-
cias et al., 2007), George and Stokes found that autistic
people reported significantly more gender dysphoric feel-
ings than non-autistic individuals. In George and Stokes’
study, however, the percentage of participants who did
not identify as cisgender was 3 times higher in the autis-
tic group than in the non-autistic group. The inclusion of
these participants in the analysis could have artificially
inflated the score of the autism group creating a signifi-
cant difference in the number of gender dysphoric feel-
ings between autistic and non-autistic people. To begin to
answer whether autistic cisgender adults have increased
gender dysphoric feelings, we examined gender dysphoric
feelings among autistic cisgender and autistic transgender
people, separately. We also examined, for the first time,
whether these groups recall limited gender-typed behavior
from childhood. On the basis of previous findings (e.g.,
Bejerot & Eriksson, 2014; Hisle-Gorman et al., 2019;
Pecora et al., 2020; Pohl et al., 2014; Walsh et al., 2018),
we made the following hypotheses: small association between ASD traits and gender vari-
ance in children from the general population (Munoz
Murakami et al., 2022), as well as an increased rate of
gender diversity among autistic children (Corbett et al.,
2022). Likewise, research among adults has shown that
autistic people report a more diverse range of gender iden-
tities than non-autistic individuals (Bejerot & Eriksson,
2014; George & Stokes, 2018b) and that they are more
likely to be planning or have transitioned (Cooper et al.,
2018). To date, only George and Stokes (2018b) have
employed a validated measure to examine gender dys-
phoric feelings in this population. Research Question 3: Do Transgender People Have
Increased Autistic‑Like Traits and Mentalizing
Difficulties? (2020) examined ASD-like traits in transgender and
gender-diverse individuals using three independent samples. In all three, transgender and gender-diverse individuals
reported significantly more ASD traits than cisgender people,
yet sex differences were not examined. Hypothesis 2a We expected autistic people who identify
either as cisgender or transgender to report significantly more
gender dysphoric feelings than non-autistic cisgender people,
autistic cisgender people to report significantly fewer gender
dysphoric feelings than non-autistic transgender individu-
als, and autistic transgender people to report significantly
more gender dysphoric feelings than non-autistic transgen-
der individuals (autistic transgender > non-autistic transgen-
der > autistic cisgender > non-autistic cisgender). f
We should note here that researchers, with the exception
of Jones et al. (2012), Murphy et al. (2020), and Warrier
et al. (2020), either did not control for (or did not collect/
report information about) the presence of autistic people in
the gender diverse samples in their studies. This is impor-
tant because research has shown a high prevalence of ASD
diagnoses in this population (e.g., Strauss et al., 2021; for
a meta-analysis see Kallitsounaki & Williams, 2022), and
as expected based on their diagnosis, autistic transgender
people show significantly more ASD traits than neurotypi-
cal transgender people (Warrier et al., 2020). Arguably,
the inclusion of autistic people in these studies could have
inflated the AQ/SRS score of gender diverse samples. Therefore, the extent to which non-autistic transgender
people have increased ASD-like traits needs further exami-
nation. Furthermore, it is still unclear whether the behavio-
ral features of ASD in transgender people are accompanied Hypothesis 2b We expected autistic people who identify
either as cisgender or transgender to recall significantly less
gender-typed behavior from childhood than non-autistic
cisgender people, autistic cisgender people to recall sig-
nificantly more gender-typed behavior from childhood than
non-autistic transgender individuals, and autistic transgender
people to recall significantly less gender-typed behavior from
childhood than non-autistic transgender individuals (autis-
tic transgender < non-autistic transgender < autistic cisgen-
der < non-autistic cisgender). 1 1 3 Archives of Sexual Behavior cisgender participants (autistic transgender < autistic cisgen-
der < non-autistic transgender < non-autistic cisgender). cisgender participants (autistic transgender < autistic cisgen-
der < non-autistic transgender < non-autistic cisgender). by the cognitive difficulties that are frequently encountered
in ASD and which arguably underpin the behavioral fea-
tures of the condition. ASD is characterized by well-established difficulties
with mentalizing (e.g., Baron-Cohen et al., 2001a; Senju
et al., 2009). Participants A total of 106 non-autistic cisgender adults (51 birth-
assigned female), 107 autistic cisgender adults (57 birth-
assigned female), 78 non-autistic transgender adults (41
birth-assigned female), and 56 autistic transgender adults
(27 birth-assigned female) took part in the current study. The mean age of participants was 31.01 years (range = 18 to
70). Participants who identified as gender nonconforming
or unknown were excluded from the study (n = 4), whereas
participants who identified as trans(masculine/male/
female) nonbinary or masculine nonbinary (n = 4) were
included in the transgender group. The number of birth-
assigned females and males did not differ significantly
between groups, χ2(3, N = 347) = 0.81, p = 0.846, φ = 0.05,
but differences in age were found, F(3, 343) = 26.99,
p < 0.001, 휂2
p = 0.19 (when participant groups were
matched for age, results of the analyses presented below
did not change substantively; see the online supplementary
material). Ninety-nine percent of participants reported
being native English speakers. All participants in the
autism groups reported having a formal diagnosis of
ASD, whereas all participants in the non-autism groups
reported that they did not have a formal diagnosis of ASD. Participants were recruited via the online crowdsourcing
platform Prolific Academic, social media platforms, and
a database of autistic individuals interested in taking part
in psychological research. All participants completed
the study online after they had given written, informed
consent and received compensation for their time. This
study was approved by the Kent Psychology Research
Ethics Committee. Another important point we should mention is that
neither in Kung’s (2020) nor Stagg and Vincent’s
(2019) study did researchers control for a possible/likely
overrepresentation of ASD diagnoses in the transgender
group. So, based on previous studies, we cannot conclude
with confidence whether non-autistic transgender people
show atypical mentalizing ability. This leaves a critical
gap in the literature that we aimed to fill by examining
ASD-like traits and mentalizing in non-autistic and
autistic transgender individuals, separately. Research Question 3: Do Transgender People Have
Increased Autistic‑Like Traits and Mentalizing
Difficulties? Mentalizing (or Theory of Mind) is the ability
to impute mental states to others (and self) in order to
interpret and predict behavior (Premack & Woodruff, 1978). Mentalizing has been proposed as one of the mechanisms
that could explain the link between ASD and gender
diversity (Glidden et al., 2016; Jacobs et al., 2014; van der
Miesen et al., 2016, 2018). To our knowledge, however,
only two studies have examined mentalizing in gender
diverse individuals. Stagg and Vincent (2019) did not find
a significant difference in Reading the Mind in the Eyes
(RMIE; Baron-Cohen et al., 2001a) performance between
cisgender and transgender people. In contrast, Kung (2020)
found that transgender individuals performed significantly
less well on the RMIE than control participants. We should
note, however, that strong conclusions cannot be drawn
from Stagg and Vincent’s (2019) study because their
sample was underpowered to detect a small (d = 0.39), but
potentially meaningful, difference between transgender and
cisgender people. Measures of Gender Self‑Concept Implicit Association Test We assessed participants’ implicit
gender self-concept using the Implicit Association Test (IAT)
described by Greenwald et al. (2002). Participants were
instructed to sort words that belonged to one of four catego-
ries using one of two possible response keys. Categories and
stimuli used in the task were (a) Self: I, me, my, mine, self;
(b) Other: they, them, their, it, other; (c) Female: woman, girl,
daughter, madam, lady, female; and (d) Male: man, boy, son,
sir, gentleman, male.i In the first block (20 trials), the “self” category label was
presented in the upper left corner of the screen and the “other”
category label in the upper right corner. Participants were
asked to sort words that belonged either to “self” or “other”
category by pressing the “a” key of a keyboard for words
related to “self” and the “l” key for words related to “other”. In the second block (20 trials), categories referred to “female”
and “male” categorization. The “female” category label was
presented in the upper left corner and the “male” category in
the upper right corner. Participants were instructed to press
the “a” key for words belonging to “female” category and the
“l” key for items belonging to “male” category. In the third
block (20 practice trials), the four categories were presented
combined (“self/female” labels: upper left corner; “other/
male” labels: upper right corner) and participants practiced
the sorting task, by pressing the key that was assigned to
each category in the preceding two blocks (i.e., “a” key for
items belonging to “self /female” categories and “l” key for
items belonging to “other/male” categories). In the fourth
block (40 experimental trials), participants completed the
first experimental condition of the combined sorting task. In the fifth block (40 trials), the “female” category label was
presented in the upper right corner and the “male” category
label in the upper left corner, subsequently the assignment
of the key for each category was reversed, compared to
the first block. Participants were instructed to press “l” for
items belonging to the “female” category and “a” for items
belonging to the “male” category. In the sixth block (20
practice trials), participants practiced the combined task
using the switched key assignments. Participants On the basis
of previous findings (Walsh et al., 2018; Warrier et al.,
2020), we made the following hypotheses: Hypothesis 3a We expected non-autistic transgender par-
ticipants to report significantly more ASD-like traits than
non-autistic cisgender people, but significantly fewer than
autistic cisgender individuals, and autistic transgender peo-
ple to report significantly more ASD-like traits than either
non-autistic transgender or autistic cisgender people (autis-
tic transgender > autistic cisgender > non-autistic transgen-
der > non-autistic cisgender). The current study was preregistered on Open Science
Framework (preregistration can be viewed here: https://osf.
io/bke5j/?view_only=795bddddc1144ef295db601982f6a1
2f). We should note, however, that none of the hypotheses
about the autistic transgender group have been included in
the preregistration. Yet, they were all made before any sta-
tistical analyses were conducted, and if preregistered, they
would be exactly the same as the ones presented in the cur-
rent article (all deviations from the preregistration, as well
as preregistered hypotheses and analyses that have not been
included in the current manuscript are reported in the online
supplementary material). Hypothesis 3b We expected non-autistic transgender individ-
uals to perform significantly less well on the mentalizing task
than non-autistic cisgender people, but significantly better
than autistic cisgender people, and autistic transgender peo-
ple to perform significantly less well on the task than autistic 1 3 Archives of Sexual Behavior (i.e., blocks 3 & 6) and the two experimental blocks
(i.e., bocks 4 & 7). When participants respond faster in
the “self-female and other-male” condition than in the
“self-male and other-female” condition, they receive a
positive D score indicating a female self-concept. When
they respond faster in the “self-male and other-female”
than in the “self-female and other-male” condition they
receive a negative D score indicating a male self-concept. When error rate in the critical blocks (i.e., practical and
experimental) exceeded 20%, participants (i.e., non-
autistic cisgender: n = 7; non-autistic transgender: n = 3;
autistic cisgender: n = 14; autistic transgender: n = 8) were
not included in the analyses (Greenwald et al., 1998; van
Well et al., 2008). Results of the analyses including all
participants, regardless of their performance on the task,
are reported in the online supplementary material. The
IAT was programmed using Inquisit Millisecond software
package 4 (2015, https://www.millisecond.com), and it
was administered using Inquisit Web Player 4.0.10. Participants After
the completion of the IAT, participants were automatically
redirected to a Qualtrics survey to complete the rest of the
tasks and questionnaires. Associations Between Autism‑Spectrum Quotient
and Performance on the Explicit and Implicit Measures
of Gender Self‑Concept In keeping with the preregistration, a series of correlation
analyses was conducted to investigate the relations between
AQ and performance on the explicit and implicit measures of
gender self-concept among non-autistic cisgender individuals
(note: scores from the explicit and implicit measures of
gender self-concept were transformed to positive values,
so that higher scores denote a stronger gender self-concept,
regardless of whether it is male or female). As predicted,
AQ score was negatively and significantly related to the
strength of the explicit gender self-concept (Hypothesis 1a),
r(104) = − 0.32, p < 0.001 (one-tailed). In keeping with the
preregistered hypotheses, AQ score was also significantly
associated with the strength of the explicit gender self-concept
(note: scores from the explicit measure were untransformed)
among both non-autistic cisgender birth-assigned males
(more ASD-like traits = weaker male self-concept) and
non-autistic cisgender birth-assigned females (more ASD-
like traits = weaker female self-concept). The results of the
analyses are included in the online supplementary material. Contrary to predictions, AQ did not correlate significantly
with the strength of the implicit gender self-concept among
non-autistic cisgender people (Hypothesis 1a), r(97) = − 0.06,
p = 0.276 (one-tailed). Results remained nonsignificant when
the analysis was performed for each birth-assigned sex
separately (see the online supplementary material). Next, we conducted a simple effects analysis of diagnostic
category within birth-assigned sex and gender identity. The
results of the analysis are illustrated in Fig. 1C and D. In line
with the preregistered Hypothesis 1c, autistic cisgender birth-
assigned males scored significantly higher on the explicit task
than non-autistic cisgender birth-assigned males, and autistic
cisgender birth-assigned females scored significantly lower on
the task than non-autistic cisgender birth-assigned females. This indicates a weaker explicit gender self-concept among
autistic cisgender participants than among non-autistic cis-
gender participants. Contrary to predictions, we found that
autistic transgender birth-assigned males scored significantly
higher on the explicit task than non-autistic transgender
birth-assigned males. This shows that autistic transgender
birth-assigned males showed a stronger explicit female self-
concept than non-autistic transgender birth-assigned males. Also unexpectedly, no difference in the strength of the explicit
gender self-concept was observed between autistic transgen-
der birth-assigned females and non-autistic transgender birth-
assigned females. Thus, autistic and non-autistic transgender
birth-assigned females displayed an explicit male gender Measures of Gender Self‑Concept That is, a standardized mean difference score (namely D)
in response latencies between the two practical blocks 1 1 3 Archives of Sexual Behavior 0.09, a significant Gender Identity × Diagnostic Category
interaction, F(1, 339) = 6.49, p = 0.011, η2
p = 0.02, and
a significant three-way interaction, F(1, 339) = 21.33,
p < 0.001, η2
p = 0.06.f the tasks and measures used in this study is provided in the
online supplementary material. the tasks and measures used in this study is provided in the
online supplementary material. Measures of Gender Self‑Concept They were instructed
to press the “a” key to categorize items that belonged either to
the “self” or to the “male” category and the “l” key for words
that belonged either to the “other” or “female” category. In
the last block (40 experimental trials), they completed the
second experimental condition of the combined sorting task. The dependent measure for the IAT was the strength
f th
t
ti
i ti
l
l t d
ith th
i Explicit Measure We assessed participants’ explicit gender
self-concept using the measure designed by Greenwald et al. (2002). Participants were asked to rate each of the six male
and six female nouns used in the IAT using a 7-point Lik-
ert scale ranging from “not at all characteristic of you” to
“extremely characteristic of you.” The measure was scored
by subtracting participants’ average score on the male nouns
from that on the female nouns. Positive scores denote a female
self-concept, and negative scores denote a male self-concept. Other Tasks and Self‑Report Measures Participants were
asked to indicate their birth-assigned sex, such as on an
original birth certificate (i.e., male or female) and their
gender identity (i.e., male, female, trans male, trans female,
gender nonconforming, or other). Participants whose birth-
assigned sex was congruent to their gender identity were
categorized as cisgender, and those whose birth-assigned
sex was incongruent to their gender identity or identified as
transgender were categorized as transgender. Participants
completed the RMIE (Baron‐Cohen et al., 2001a), which
is a widely used measure of adult mentalizing, the AQ-50
(Baron-Cohen et al., 2001b) that measures ASD-like traits
(scores ≥ 26 denote clinically significant levels of ASD-like
traits), the GIDYQ (Deogracias et al., 2007), which is a reli-
able self-report measure that taps gender identity and gender
dysphoria (scores ≤ 3 denote clinically significant levels of
gender dysphoria), and the 18 items of the Recalled Child-
hood Gender Identity/Gender Role Questionnaire (RCGI;
Zucker et al., 2006) that assess gender role behavior and
gender identity. Please note that a detailed description of The dependent measure for the IAT was the strength
of the automatic associations, calculated with the scoring
algorithm recommended by Greenwald et al. (2003). Results p
Breaking down the three-way interaction, a simple effects
analysis of birth-assigned sex within gender identity and
diagnostic category indicated that in accordance with the
preregistered Hypothesis 1b, the explicit measure of gender
self-concept was sensitive to gender identity differences. Results of the analysis are illustrated in Fig. 1A and B. Spe-
cifically, we found that among non-autistic and autistic cisgen-
der individuals, birth-assigned females scored significantly
higher than birth-assigned males (note: mean scores of birth-
assigned females were significantly above zero, and mean
scores of birth-assigned males were significantly below zero,
all ps < 0.001, one-tailed). This indicates that birth-assigned
females who identify as females showed an explicit female
self-concept and birth-assigned males who identify as males
showed an explicit male self-concept. The opposite pattern
was observed among non-autistic and autistic transgender
individuals, with birth-assigned females scoring significantly
lower than birth-assigned males (note: mean scores of birth-
assigned females were significantly below zero, and mean
scores of birth-assigned males were significantly above zero,
all ps < 0.001, one-tailed). This indicates that birth-assigned
females who identify as males showed an explicit male self-
concept in line with their experienced (rather than birth-
assigned) gender, whereas birth-assigned males who identify
as females showed an explicit female self-concept in line with
their experienced (rather than birth-assigned) gender. These
results were entirely expected.f Gender‑Related Cognition Performance on the Explicit Measure of Gender
Self‑Concept Specifically, we found
that among non-autistic and autistic cisgender individu-
als, birth-assigned females scored significantly higher than
birth-assigned males (note: mean scores of birth-assigned
females were significantly above zero (non-autistic
p < 0.001, one-tailed; autistic p = 0.003, one-tailed), and
mean scores of birth-assigned males were significantly
below zero (all ps < 0.001, one-tailed). This indicates that
birth-assigned females who identify as females showed an
implicit female self-concept, whereas birth-assigned males
who identify as males showed an implicit male self-con-
cept. The opposite pattern was observed among non-autis-
tic and autistic transgender individuals, with birth-assigned
females scoring significantly lower than birth-assigned
males (note: mean scores of birth-assigned females were
significantly below zero, and mean scores of birth-assigned
males were significantly above zero, all ps < 0.001, one-
tailed). This indicates that birth-assigned females who
identify as males showed an implicit male self-concept
in line with their experienced (rather than birth-assigned)
gender, and birth-assigned males who identify as females
showed an implicit female self-concept in line with their self-concept that was in keeping with their experienced gen-
der, rather than birth-assigned gender, to the same degree. Performance on the Explicit Measure of Gender
Self‑Concept A 2 (birth-assigned sex: male/female) × 2 (diagnostic cate-
gory: non-autistic/autistic) × 2 (gender identity: cisgender/
transgender) ANOVA was conducted on participant scores
from the explicit measure of gender self-concept. Nonsig-
nificant main effects were detected for birth-assigned sex,
F(1, 339) = 2.18, p = 0.140, η2
p = 0.01, gender identity, F(1,
339) = 0.05, p = 0.829, η2
p = 0.00, and diagnostic category,
F(1, 339) = 0.14, p = 0.707, η2
p = 0.00. Nonetheless, the
analysis revealed a significant Birth-Assigned Sex × Gen-
der Identity interaction, F(1, 339) = 2843.37, p < 0.001,
η2
p = 0.89, a significant Birth-Assigned Sex × Diagnostic
Category interaction, F(1, 339) = 34.46, p < 0.001, η2
p = 1 3 3 Archives of Sexual Behavior Fig. 1 Performance on the
explicit measure of gender
self-concept as a function of
birth-assigned sex and diagnos-
tic category within cisgender
and transgender participants
Note. Birth-assigned males:
Non-ASD cisgender n = 55;
ASD cisgender n = 50; Non-
ASD transgender n = 37; ASD
transgender n = 29. Birth-
assigned females: Non-ASD
cisgender n = 51; ASD cisgen-
der n = 57; Non-ASD transgen-
der n = 41; ASD transgender
n = 27. For ps < 0.001, 휂2
p ≥
0.06; for p < 0.05, 휂2
p = 0.01; for
p > 0.05, 휂2
p = 0.00. *p < 0.05
(one-tailed). *** p < 0.001
(one-tailed) Fig. 1 Performance on the
explicit measure of gender
self-concept as a function of
birth-assigned sex and diagnos-
tic category within cisgender
and transgender participants
Note. Birth-assigned males:
Non-ASD cisgender n = 55;
ASD cisgender n = 50; Non-
ASD transgender n = 37; ASD
transgender n = 29. Birth-
assigned females: Non-ASD
cisgender n = 51; ASD cisgen-
der n = 57; Non-ASD transgen-
der n = 41; ASD transgender
n = 27. For ps < 0.001, 휂2
p ≥
0.06; for p < 0.05, 휂2
p = 0.01; for
p > 0.05, 휂2
p = 0.00. *p < 0.05
(one-tailed). *** p < 0.001
(one-tailed) with the preregistered Hypothesis 1b, the IAT was sensi-
tive to gender identity differences. Results of the analysis
are illustrated in Fig. 2A and B. Performance on the Implicit Measure of Gender
Self‑Concept A 2 (birth-assigned sex: male/female) × 2 (diagnostic
category: non-autistic/autistic) × 2 (gender identity:
cisgender/transgender) ANOVA was conducted on participant
scores from the IAT. Significant main effects were detected
for birth-assigned sex, F(1, 304) = 6.41, p = 0.012, η2
p = 0.02,
gender identity, F(1, 304) = 7.64, p = 0.006, η2
p = 0.03, and
diagnostic category, F(1, 304) = 13.83, p < 0.001, η2
p = 0.04. The analysis also yielded a significant Birth-Assigned Sex ×
Gender Identity interaction, F(1, 304) = 302.40, p < 0.001,
η2
p = 0.50 and a significant Gender Identity × Diagnostic
Category interaction, F(1, 304) = 4.98, p = 0.026, η2
p = 0.02. Neither the Birth-Assigned Sex × Diagnostic Category
interaction, F(1, 304) = 0.15, p = 0.701, η2
p = 0.00, nor the
three-way interaction, F(1, 304) = 2.80, p = 0.095, η2
p = 0.01
were significant. i
Breaking down the three-way interaction, a simple
effects analysis of birth-assigned sex within gender iden-
tity and diagnostic category indicated that in accordance 1 Archives of Sexual Behavior Fig. 2 Performance on the
implicit measure of gender
self-concept as a function of
birth-assigned sex and diagnos-
tic category within cisgender
and transgender participants
Note. Birth-assigned males:
Non-ASD cisgender n = 50;
ASD cisgender n = 43; Non-
ASD transgender n = 34; ASD
transgender n = 26. Birth-
assigned females: Non-ASD
cisgender n = 49; ASD cisgen-
der n = 48; Non-ASD transgen-
der n = 40; ASD transgender
n = 22. For ps < 0.001, 휂2
p ≥
0.07; for p < 0.05, 휂2
p = 0.02; for
ps > 0.05, 휂2
p ≤ 0.01 *p < 0.05
(one-tailed). *** p < 0.001
(one-tailed) Performance on Reading the Mind in the Eyes
and Self‑Report Measures Performance on Reading the Mind in the Eyes
and Self‑Report Measures experienced (rather than birth-assigned) gender. These
results were entirely expected.f Next, we conducted a simple effects analysis of Diagnos-
tic Category within Birth-assigned Sex and Gender Identity. The results of the analysis are illustrated in Fig. 2C and
D. In line with the preregistered Hypothesis 1c, we found
that autistic cisgender birth-assigned females achieved a
significantly lower D score on the IAT than non-autistic cis-
gender birth-assigned females, indicating a weaker implicit
female self-concept. Contrary to predictions, we found that
autistic cisgender birth-assigned males achieved a signifi-
cantly lower D score on the IAT than non-autistic cisgender
birth-assigned males, indicating a stronger implicit male
self-concept. Also unexpectedly, a nonsignificant difference
in the strength of implicit gender self-concept was found
between autistic and non-autistic transgender individuals. Both autistic and non-autistic transgender adults displayed
an implicit gender self-concept that was in keeping with
their experienced gender, rather than birth-assigned gender,
to the same degree. Table 1 shows descriptive statistics for participant scores on
GIDYQ, RCGI, AQ, and RMIE, and Table 2 shows the results
of a series of ANOVAs we conducted on these scores. Gender Dysphoric Feelings Hence, their data has not been included in the analysis
Groups
Age
RMIE
GIDYQ a,b
RCGI a,c
AQ
M (SD)
M (SD)
M (SD)
M (SD)
M (SD)
Non-autistic cis
37.10 (12.89)
26.09 (4.65)
4.80 (0.20)
3.94 (0.52)
19.98 (7.42)
Non-autistic trans
26.51 (9.02)
26.59 (3.77)
2.16 (0.32)
2.63 (0.63)
24.55 (9.24)
Autistic cis
31.55 (7.86)
19.61 (7.80)
4.18 (0.71)
3.52 (0.63)
31.45 (7.21)
Autistic trans
24.73 (6.85)
24.66 (4.70)
2.23 (0.45)
2.58 (0.64)
35.11 (6.70) Cis Cisgender, Trans Transgender, Age Measured in years, RMIE Reading the Mind in the Eyes (0–36),
GIDYQ Gender Identity/Gender Dysphoria Questionnaire (1–5), RCGI Recalled Childhood Gender
Identity/Gender Role Questionnaire (1–5), AQ Autism-Spectrum Quotient (0–50) a One non-autistic cisgender birth-assigned male completed the female version of the GIDYQ and RCGI,
and one autistic cisgender birth-assigned female completed the male version of the GIDYQ and RCGI. Hence, their data has not been included in the analysis a One non-autistic cisgender birth-assigned male completed the female version of the GIDYQ and RCGI
and one autistic cisgender birth-assigned female completed the male version of the GIDYQ and RCGI
Hence, their data has not been included in the analysis b Low scores = more gender dysphoria. Gender Dysphoric Feelings A 2 (birth-assigned sex: male/female) × 2 (diagnostic cat-
egory: non-autistic/autistic) × 2 (gender identity: cisgender/
transgender) ANOVA was conducted on GIDYQ scores. Significant main effects were detected for birth-assigned
sex, such that birth-assigned females (marginal M = 3.25,
SE = 0.04) reported significantly more gender dysphoric
feelings than birth-assigned males (marginal M = 3.44,
SE = 0.04), gender identity, such that transgender individuals
(marginal M = 2.20, SE = 0.04) reported significantly more
gender dysphoric feelings than cisgender people (marginal
M = 4.49, SE = 0.03), and diagnostic category, such that 1 3 3 Archives of Sexual Behavior Table 1 Participant
characteristics and mean (SD)
performance on Reading the
Mind in the Eye and self-report
measures
Cis Cisgender, Trans Transgender, Age Measured in years, RMIE Reading the Mind in the Eyes (0–36),
GIDYQ Gender Identity/Gender Dysphoria Questionnaire (1–5), RCGI Recalled Childhood Gender
Identity/Gender Role Questionnaire (1–5), AQ Autism-Spectrum Quotient (0–50)
a One non-autistic cisgender birth-assigned male completed the female version of the GIDYQ and RCGI,
and one autistic cisgender birth-assigned female completed the male version of the GIDYQ and RCGI. Hence, their data has not been included in the analysis
b Low scores = more gender dysphoria. c Low scores = less recalled gender-typed behavior from childhood
Groups
Age
RMIE
GIDYQ a,b
RCGI a,c
AQ
M (SD)
M (SD)
M (SD)
M (SD)
M (SD)
Non-autistic cis
37.10 (12.89)
26.09 (4.65)
4.80 (0.20)
3.94 (0.52)
19.98 (7.42)
Non-autistic trans
26.51 (9.02)
26.59 (3.77)
2.16 (0.32)
2.63 (0.63)
24.55 (9.24)
Autistic cis
31.55 (7.86)
19.61 (7.80)
4.18 (0.71)
3.52 (0.63)
31.45 (7.21)
Autistic trans
24.73 (6.85)
24.66 (4.70)
2.23 (0.45)
2.58 (0.64)
35.11 (6.70) Cis Cisgender, Trans Transgender, Age Measured in years, RMIE Reading the Mind in the Eyes (0–36),
GIDYQ Gender Identity/Gender Dysphoria Questionnaire (1–5), RCGI Recalled Childhood Gender
Identity/Gender Role Questionnaire (1–5), AQ Autism-Spectrum Quotient (0–50)
a One non-autistic cisgender birth-assigned male completed the female version of the GIDYQ and RCGI,
and one autistic cisgender birth-assigned female completed the male version of the GIDYQ and RCGI. Questionnaire, AQ Autism-Spectrum Quotient, RMIE Reading the Mind in the Eyes Sex Sex-assigned at birth, GIDYQ Gender Identity/Gender Dysphoria Questionnaire, RCGI Recalled C Recalled Gender‑Typed Behavior from Childhood A 2 (birth-assigned sex: male/female) × 2 (diagnostic cat-
egory: non-autistic/autistic) × 2 (gender identity: cisgender/
transgender) ANOVA was conducted on RCGI scores. Signif-
icant main effects were detected for birth-assigned sex, such
that birth-assigned females (marginal M = 2.89, SE = 0.04)
recalled significantly less gender-typed behavior from
childhood than birth-assigned males (marginal M = 3.46,
SE = 0.04), gender identity, such that transgender partici-
pants (marginal M = 2.61, SE = 0.05) recalled significantly
less gender-typed behavior from childhood than cisgender
participants (marginal M = 3.73, SE = 0.04), and diagnostic To test Hypothesis 2a, the significant Gender Identity ×
Diagnostic Category interaction was treated as a 4-level vari-
able and a series of planned t-tests was conducted. Results of
the analyses are presented in Table 3. As predicted, autistic
cisgender people reported significantly more gender dys-
phoric feelings than non-autistic cisgender people, but sig-
nificantly fewer than non-autistic and autistic transgender
participants. Contrary to predictions, there was no significant
difference in GIDYQ score between non-autistic and autistic Cis Cisgender, Trans Transgender, GIDYQ Gender Identity/Gender Dysphoria Questionnaire, RCGI
Recalled Childhood Gender Identity/Gender Role Questionnaire, AQ Autism-spectrum Quotient, 95% CI
95% Confidence Intervals
a Please note that when groups were matched for age the p value was less than 0.01 (one-tailed) and the size
of the between-group difference was medium (i.e., d = 0.54). *p < 0.05. **p < 0.01. Gender Dysphoric Feelings c Low scores = less recalled gender-typed behavior from childhood Table 2 2 (Birth-assigned Sex: Male/Female) × 2 (Diagnostic Category: Non-ASD/ASD) × 2 (Gender Identity: Cisgender/Transgender) ANOVA
results
Sex Sex-assigned at birth, GIDYQ Gender Identity/Gender Dysphoria Questionnaire, RCGI Recalled Childhood Gender Identity/Gender Role
Q
i
i
AQ A i
S
Q
i
RMIE R
di
h Mi d i
h E
Measure
Effect
F
p
휂2
p
Direction of main effects
GIDYQ
Sex
12.55
< 0.001
0.04
Birth-assigned females < Birth-assigned males
Gender identity
1947.98
< 0.001
0.85
Transgender < Cisgender
Diagnostic category
28.73
< 0.001
0.08
Autistic < Non-autistic
Gender identity × Diagnostic category
41.11
< 0.001
0.11
Sex × Gender identity
3.35
0.068
0.01
Sex × Diagnostic category
0.78
0.378
0.00
Sex × Gender identity × Diagnostic category
0.10
0.756
0.00
RCGI
Sex
92.67
< 0.001
0.22
Birth-assigned females < Birth-assigned males
Gender identity
361.98
< 0.001
0.52
Transgender < Cisgender
Diagnostic category
16.63
< 0.001
0.05
Autistic < Non-autistic
Gender identity × Diagnostic category
7.37
0.007
0.02
Sex × Gender identity
1.10
0.295
0.00
Sex × Diagnostic category
0.83
0.364
0.00
Sex × Gender identity × Diagnostic category
0.99
0.322
0.00
AQ
Sex
3.32
0.069
0.01
Birth-assigned females = Birth-assigned males
Gender identity
22.79
< 0.001
0.06
Transgender > Cisgender
Diagnostic category
167.92
< 0.001
0.33
Autistic > Non-autistic
Gender identity × Diagnostic category
0.21
0.645
0.00
Sex × Gender identity
2.67
0.103
0.01
Sex × Diagnostic category
0.26
0.608
0.00
Sex × Gender identity × Diagnostic category
0.34
0.563
0.00
RMIE
Sex
7.50
0.006
0.02
Birth-assigned females < Birth-assigned males
Measure
Effect
F
p
휂2
p
Direction of the main effects
Gender identity
19.02
< 0.001
0.05
Transgender > Cisgender
Diagnostic category
45.40
< 0.001
0.12
Autistic < Non-autistic
Gender identity × Diagnostic category
12.63
< 0.001
0.04
Sex × Gender identity
2.33
0.128
0.01
Sex × Diagnostic category
7.34
0.007
0.02
Sex × Gender identity × Diagnostic category
1.59
0.209
0.01 1 Archives of Sexual Behavior transgender individuals (hence, autistic transgender = non-
autistic transgender < autistic cisgender < non-autistic
cisgender). autistic people (marginal M = 3.21, SE = 0.04) reported sig-
nificantly more gender dysphoric feelings than non-autistic
individuals (marginal M = 3.48, SE = 0.04). The analysis also
revealed a significant Gender Identity × Diagnostic Cate-
gory interaction. Gender Dysphoric Feelings The Birth-Assigned Sex × Gender Identity
interaction was nonsignificant, as were the Birth-Assigned
Sex × Diagnostic Category interaction and the three-way
interaction.i i
a Please note that when groups were matched for age the p value was less than 0.01 (one-tailed) and the size
of the between-group difference was medium (i.e., d = 0.54). *p < 0.05. **p < 0.01. ***p < 0.001 Cis Cisgender, Trans Transgender, GIDYQ Gender Identity/Gender Dysphoria Questionnaire, RCGI
Recalled Childhood Gender Identity/Gender Role Questionnaire, AQ Autism-spectrum Quotient, 95% CI
95% Confidence Intervals Recalled Gender‑Typed Behavior from Childhood ***p < 0.001
Measure
t-tests
Cohen’s d
95% CI
GIDYQ
Non-autistic cis > Non-autistic trans ***
10.18
[9.09, 11.27]
Non-autistic cis > Autistic cis ***
1.18
[0.88, 1.47]
Non-autistic cis > Autistic trans ***
8.37
[7.39, 9.34]
Non-autistic trans < Autistic cis ***
− 3.47
[− 3.93, − 3.01]
Non-autistic trans = Autistic trans
− 0.18
[− 0.52, 0.17]
Autistic cis > Autistic trans ***
3.08
[2.61, 3.54]
RCGI
Non-autistic cis > Non-autistic trans ***
2.30
[1.92, 2.67]
Non-autistic cis > Autistic cis ***
0.73
[0.45, 1.01]
Non-autistic cis > Autistic trans ***
2.42
[2.00, 2.83]
Non-autistic trans < Autistic cis ***
− 1.41
[− 1.74, − 1.08]
Non-autistic trans = Autistic trans
0.08
[− 0.26, 0.43]
Autistic cis > Autistic trans ***
1.49
[1.13, 1.85]
AQ
Non-autistic cis < Non-autistic trans ***
− 0.56
[− 0.85, − 0.26]
Non-autistic cis < Autistic cis ***
− 1.57
[− 1.87, − 1.26]
Non-autistic cis < Autistic trans ***
− 2.11
[− 2.50, − 1.71]
Non-autistic trans < Autistic cis ***
− 0.85
[− 1.15, − 0.54]
Non-autistic trans < Autistic trans ***
− 1.28
[− 1.65, − 0.90]
Autistic cis < Autistic trans **
− 0.52
[− 0.85, − 0.19]
RMIE
Non-autistic cis = Non-autistic trans
− 0.12
[− 0.41, 0.18]
Non-autistic cis > Autistic cis ***
1.01
[0.72, 1.29]
Non-autistic cis > Autistic trans * a
0.31
[− 0.02, 0.63]
Non-autistic trans > Autistic cis ***
1.09
[0.77, 1.40]
Non-autistic trans > Autistic trans **
0.46
[0.11, 0.81]
Autistic cis < Autistic trans ***
− 0.73
[− 1.06, − 0.40] Table 3 Planned comparisons
between groups 1 3 Archives of Sexual Behavior category, such that autistic participants (marginal M = 3.05,
SE = 0.04) recalled significantly less gender-typed behavior
than non-autistic participants (marginal M = 3.29, SE = 0.04). The analysis also revealed a significant Gender Identity ×
Diagnostic Category interaction. The Birth-Assigned Sex ×
Gender Identity interaction was nonsignificant, as were the
Birth-Assigned Sex × Diagnostic Category interaction and
the three-way interaction.i cisgender/transgender) ANOVA was conducted on RMIE
scores. Recalled Gender‑Typed Behavior from Childhood Significant main effects were detected for birth-
assigned Sex, such that birth-assigned males (marginal
M = 25.11, SE = 0.44) performed significantly better on
the task than birth-assigned females (marginal M = 23.43,
SE = 0.43), gender identity, such that transgender participants
(marginal M = 25.61, SE = 0.48) performed significantly
better on the task than cisgender participants (marginal
M = 22.93, SE = 0.38), and diagnostic category, such that
non-autistic participants (marginal M = 26.34, SE = 0.41)
performed significantly better on the task than autistic
participants (marginal M = 22.20, SE = 0.46). The analysis
also revealed a significant Birth-Assigned Sex × Diagnostic
Category interaction and a significant Gender Identity
× Diagnostic Category interaction. Neither the Birth-
Assigned Sex × Gender Identity interaction nor the three-way
interaction were significant.i To test Hypothesis 2b, the significant Gender Identity
× Diagnostic Category interaction was treated as a 4-level
variable and a series of planned t-tests was conducted. Results of the analyses are presented in Table 3. As predicted,
autistic cisgender people recalled significantly less gender-
typed behavior from childhood than non-autistic cisgender
people, but significantly more than non-autistic and autistic
transgender participants. Contrary to predictions, there
was no significant difference in RCGI score between
non-autistic and autistic transgender individuals (hence,
autistic transgender = non-autistic transgender > autistic
cisgender > non-autistic cisgender). i
To test Hypothesis 3b, the significant Gender Identity
× Diagnostic Category interaction was treated as a 4-level
variable and a series of planned t-tests was conducted. Results of the analyses are presented in Table 3. In contrast
to predictions, no differences emerged in RMIE task
performance between non-autistic transgender and non-
autistic cisgender people, but as expected, both groups
performed significantly better than autistic cisgender people. In keeping with predictions, autistic transgender participants
scored significantly lower on the task than both non-autistic
cisgender and non-autistic transgender people, but contrary
to predictions, they scored significantly higher than autistic
cisgender people (hence, autistic cisgender < autistic
transgender < non-autistic transgender = non-autistic
cisgender). Discussion In the current study we examined three core aspects of the
link between ASD and gender diversity as a whole, aiming to
acquire a more holistic understanding of intersectional identi-
ties (autistic + gender diverse). We found that relative to non-
autistic cisgender people, autistic cisgender people showed a
less strong explicit identification with gender groups that cor-
responded to their experienced gender (and that autistic cis-
gender birth-assigned females, but not males, showed a less
strong implicit identification, too). This was accompanied by
fewer memories of gender-typed behavior in childhood and
increased (albeit not clinically significant) levels of current
gender dysphoric feelings. In contrast, autistic transgender
people showed a strong identification with gender groups
that matched their experienced gender, and their levels of
childhood gender-typed behavior and gender dysphoria were
equivalent to levels reported by non-autistic transgender To examine Hypothesis 3a, a series of planned t-tests
was conducted. Results of the analyses are presented in
Table 3. As predicted, non-autistic transgender participants
reported significantly more ASD-like traits than non-
autistic cisgender people, but significantly fewer than
autistic cisgender individuals. We also found that autistic
transgender individuals reported significantly more
ASD-like traits than autistic cisgender people (hence,
autistic transgender > autistic cisgender > non-autistic
transgender > non-autistic cisgender). Autism‑like Traits A 2 (birth-assigned sex: male/female) × 2 (diagnostic
category: non-autistic/autistic) × 2 (gender identity:
cisgender/transgender) ANOVA was conducted on AQ scores. Significant main effects were detected for gender identity,
such that transgender participants (marginal M = 29.82,
SE = 0.67) reported significantly more ASD-like traits than
cisgender participants (marginal M = 25.74, SE = 0.53), and
diagnostic category, such that autistic participants (marginal
M = 33.32, SE = 0.63) reported significantly more ASD-like
traits than non-autistic participants (marginal M = 22.24,
SE = 0.57). The main effect of birth-assigned sex was
nonsignificant, as were the Birth-Assigned Sex × Gender
Identity interaction, the Birth-Assigned Sex × Diagnostic
Category interaction, the Gender Identity × Diagnostic
Category interaction, and the three-way interaction. Implicit and Explicit Gender‑Related Cognition
in Autism Spectrum Disorder The first aim of the current study was to examine whether
ASD affects gender-related cognition. To do so, we first
investigated the relation between ASD-like traits, on the one
hand, and the strength of explicit and implicit gender self-
concept, on the other hand, among non-autistic cisgender
people. The approach followed was taken by Kallitsounaki
and Williams (2020a) to enable our attempt to replicate
(conceptually) their findings. In keeping with predictions,
AQ score was negatively and significantly associated with the
strength of the explicit gender self-concept. Results indicate
that non-autistic cisgender people with elevated ASD-like
traits have a weaker propensity to explicitly self-identify with
gender groups. This finding adds to recent evidence provided
by Kallitsounaki and Williams (2020a) that cisgender people
with high ASD-like traits display a weaker inclination to
explicitly identify with personality traits that stereotypically
characterize either females or males. As levels of ASD-like
traits increase, so too may be reporting the experience of a
gender self-concept that it is not strongly associated with
either feminine/masculine attributes or a collective gender
identity. One possible explanation for this association is
that people with high ASD traits might feel less obliged
to conform to the societal expectations of how one’s own
gender should be presented at a behavioral level. This could
be attributed to a reduced experience of self-conscious
emotions (e.g., pride and guilt; Davidson et al., 2017). Of
course, further research is required to test this hypothesis. Next, we aimed to extend Kallitsounaki and Williams’
(2020a) findings further by investigating, for the first time,
gender-related cognition in autistic cisgender and transgender
people, using an explicit and implicit measure of gender self-
concept. As expected, both measures tapped the experienced
gender of participants. Specifically, among cisgender
individuals, either non-autistic or autistic, birth-assigned
females showed an explicit and implicit female self-concept
and birth-assigned males showed an explicit and implicit
male self-concept. Whereas, among transgender individuals,
either non-autistic or autistic, birth-assigned females who
identify as males showed an explicit and implicit male self-
concept and birth-assigned males who identify as females
showed an explicit and implicit female self-concept. To our knowledge, this is the first study to investigate
implicit gender self-concept in transgender adults and
found that non-autistic and autistic transgender adults
perceive themselves explicitly and implicitly in terms of
their experienced gender. Olson et al. Mentalizing Ability A 2 (birth-assigned sex: male/female) × 2 (diagnostic
category: non-autistic/autistic) × 2 (gender identity: 1 Archives of Sexual Behavior people. Lastly, the latter group presented elevated ASD-like
traits. However, these traits were not accompanied by chal-
lenges with mentalizing. Below follows a more extensive
discussion of the findings. derived from different theoretical and research backgrounds
(Wood & Eagly, 2015). This should be taken into account in
future gender-related studies that use an IAT. It could be the
case that non-autistic individuals with high ASD-like traits
implicitly identify with gender-stereotypical traits to a lesser
degree than non-autistic individuals with low ASD-like traits,
but nonetheless, show no difference in implicit identification
with gender groups. Implicit and Explicit Gender‑Related Cognition
in Autism Spectrum Disorder Furthermore, on the basis of previous findings within the 1 3 3 Archives of Sexual Behavior general population (Kallitsounaki & Williams, 2020a), we
expected autistic cisgender females to show a significantly
lower score on the implicit measure of gender self-concept
than non-autistic cisgender females. Indeed, our hypothesis
was confirmed. This important finding is the first of its
kind, to our knowledge, and suggests that autistic cisgender
females have a weaker inclination to incorporate into their
self-concept a collective gender concept that matches their
birth-assigned sex. We also predicted that autistic cisgender
males would show a significantly lower score on the implicit
measure of gender self-concept than non-autistic cisgender
males. Unexpectedly, however, autistic cisgender males
displayed a significantly higher score on the implicit measure
of gender self-concept than non-autistic cisgender males,
indicating a stronger inclination to incorporate into their self-
concept a collective gender concept that matches their birth-
assigned sex. Thus, there is an important effect of sex here. associated with their birth-assigned sex, but the strength of
the identification was weaker only among birth-assigned
females. In contrast, autistic transgender and non-autistic
transgender people identified with their experienced gender
at least to the same degree, both explicitly and implicitly. On this basis, it could be argued that these results provide
preliminary evidence of a selective influence of ASD in the
consolidation of a collective gender self-concept that matches
people’s birth-assigned sex. To put it simply, ASD seems to hinder the explicit (and
implicit among birth-assigned females) identification of
people only with the gender groups associated with their
birth-assigned sex. The explicit and implicit identification
of autistic transgender people with the gender groups of their
experienced gender seems to be unaffected by ASD. It is
possible that the formation and consolidation of a gender
self-concept that does not correspond to birth-assigned sex
might be achieved through trajectories and mechanisms
that are different from the ones followed when a gender
self-concept corresponds to birth-assigned sex. To get a
better understanding of the findings from the autistic adult
population (cisgender and transgender), we believe it is
essential to know how gender self-concepts develops in
autistic children. Given that research on this topic is almost
nonexistent, future research will be needed to elucidate
whether the development of gender self-concept follows the
same cognitive and developmental trajectories in autistic
and non-autistic children (van Schalkwyk et al., 2015). Implicit and Explicit Gender‑Related Cognition
in Autism Spectrum Disorder (2015) rightly, in our
view, highlighted that “the IAT should not be seen as a lie
detector test” (p. 468). Taken together, however, results from
the explicit and implicit task, it can be argued that the current
study might provide counter evidence to the hypothesis
that symptoms of gender dysphoria in ASD (e.g., cross-
dressing) reflect more an obsession that arises from autistic
people’s inherent predisposition toward unusual interests and
preoccupations than a “genuine” mismatch between their
experienced gender and their birth-assigned sex (Parkinson,
2014; Tateno et al., 2008). Furthermore, results indicate
that autistic people (cisgender and transgender) are able
to formulate a gender self-concept, regardless of whether
it matches their birth-assigned sex. It is unclear, however,
whether autistic and non-autistic people identify with their
experienced gender to the same degree. To answer to this
question, we first compared the strength of explicit and
implicit gender self-concept between autistic and non-autistic
cisgender people. q
yp
Contrary to predictions, the number of self-reported
ASD-like traits was not significantly associated with the
strength of the implicit gender self-concept. This finding is
inconsistent with that of Kallitsounaki and Williams (2020a)
who observed a significant association between number of
ASD-like traits and the strength of the implicit gender self-
concept. Thus, contrary to the results of the current study,
Kallitsounaki and Williams found the higher the ASD-like
traits among cisgender people the weaker the automatic
identification of self with either masculine or feminine
personality traits. One possible explanation for why the
current study failed to replicate the results of the original
study is that in this study we used an IAT that taps implicit
self-identification with gender groups, whereas Kallitsounaki
and Williams (2020a) used an IAT that taps implicit self-
identification with gender-stereotypical personality traits. Although both approaches have been traditionally used in
gender identity research, they are not equivalent, as both have As predicted, autistic cisgender individuals (birth-assigned
males and females) showed a weaker explicit identification
with the gender groups associated with their birth-assigned
sex than non-autistic cisgender people. This was expected,
given previous findings of lower explicit gender identification
in autistic people (Cooper et al., 2018) and people with
increased ASD-like traits (Kallitsounaki & Williams, 2020a). Implicit and Explicit Gender‑Related Cognition
in Autism Spectrum Disorder The
second overarching aim of this study was to investigate the
extent to which the strength of gender-group identification
autistic people show is in line with their current gender
dysphoric feelings and recalled childhood gender-typed
behavior. f
Autistic cisgender females show weaker implicit and
explicit identification with female gender groups than non-
autistic cisgender females. In contrast, autistic cisgender males
show weaker explicit, yet stronger implicit identification
with male gender groups than non-autistic cisgender males. The observed mismatch between their explicit and implicit
experience of male self-concept among autistic cisgender
males might reflect a difference in self-awareness. The idea
that there are differences in self-awareness between autistic
and non-autistic people has been expressed by many (e.g.,
Frith & Happé, 1999; Hobson, 1990; Williams, 2010), but
to our knowledge, sex differences in self-awareness have not
been examined in the autistic population and results from
studies in the general population remain inconclusive (e.g.,
Jonsson & Allwood, 2003; Lemieux et al., 2019; Prentice &
Murphy, 2022). Next, we compared the strength of explicit and implicit
gender self-concept between autistic transgender and non-
autistic transgender individuals. Contrary to predictions,
autistic transgender and non-autistic transgender birth-
assigned females identified explicitly with the gender groups
associated with their experienced gender to the same degree,
and autistic transgender birth-assigned males identified
explicitly with the gender groups of their experienced
gender more strongly than non-autistic transgender birth-
assigned males. Also unexpectedly, the performance of
autistic transgender individuals (birth-assigned males and
females) on the implicit measure of gender self-concept did
not differ significantly from the performance of non-autistic
transgender individuals. Both groups identified implicitly
with their experienced gender, rather than their birth-
assigned gender, to the same degree. Gender Dysphoric Feelings and Recalled
Gender‑Typed Behavior in Autism Spectrum
Disorder This is in
line with recent findings that autistic people report a more
diverse range of gender identities and are more likely to be
gender diverse and to have or be planning a gender transition
than non-autistic people (Bejerot & Eriksson, 2014; Cooper
et al., 2018; George & Stokes, 2018b). This also supports
the hypothesis that there is link between ASD and gender
diversity (e.g., Strang et al., 2018a). Nonetheless, we should
stress that the mechanisms that could underpin this link
are still unclear and further research is required. Given
the risks involved in this type of research such endeavors
might be best supported by community involvement (e.g.,
community-based participatory approaches to help direct
and contextualize such research). Epidemiological studies
are also required to get an estimate of the size of this link. To
date, the only study of the prevalence of gender dysphoria
diagnosis in the autistic population has been conducted
among children (Hisle-Gorman et al., 2019). The hypothesis
of a link between ASD and gender diversity has also been
supported by evidence of increased ASD traits in gender
diverse people (Kallitsounaki & Williams, 2022). Therefore,
the last overarching aim of this study was to examine whether
transgender individuals have increased ASD-like traits and/
or difficulties in mentalizing ability. We should highlight here that it remains debatable whether
ASD-like traits observed among gender diverse people tap
“true” ASD characteristics (Fortunato et al., 2022; Turban,
2018; Turban & van Schalkwyk, 2018). It has been suggested
that the psychological impact of stigma, marginalization,
and rejection that transgender people frequently experience
increase their liability to develop features that “mimic” ASD
characteristics (Fortunato et al., 2022; Turban, 2018; Turban
& van Schalkwyk, 2018). To investigate this hypothesis we
examined whether the behavioral features of ASD observed
among transgender individuals (non-autistic and autistic)
were accompanied by (traditionally-observed) difficulties at
the cognitive level. If ASD-like traits among non-autistic and
autistic transgender people reflected true ASD characteristics,
then these groups should show the mentalizing difficulty that
is frequently observed in ASD. Interestingly, this was not the case. Non-autistic transgen-
der people’s performance on the mentalizing task was equiva-
lent to the performance of non-autistic cisgender individuals. This is out of keeping with Kung (2020) who found that
transgender males had significantly poorer mentalizing abil-
ity than cisgender females and transgender females poorer
mentalizing ability than cisgender males. Gender Dysphoric Feelings and Recalled
Gender‑Typed Behavior in Autism Spectrum
Disorder We first examined whether autistic transgender people
report increased current gender dysphoric feelings and
recall limited gender-typed behavior from childhood. To
our knowledge, this is the first study that attempted to
answer this question. We found that in accordance to the
mismatch autistic transgender people expressed between
their birth-assigned sex and their experienced gender, they
reported clinically significant levels of gender dysphoria
and they recalled limited gender-typed behavior from
childhood. This indicates that autistic transgender and non-
autistic transgender people feel an extreme distress of their
body, anatomy, and function to the same degree and that
feelings related to gender diversity had an early onset in
both groups. Taken together, these findings provide further
support to the clinical recommendation that adequate In sum, autistic cisgender people were able to identify
explicitly with the gender groups of their birth-assigned sex,
yet they identified less strongly than did non-autistic cisgender
people. They also identified implicitly with the gender groups 1 3 3 Archives of Sexual Behavior 2018; Warrier et al., 2020), but further research is required to
understand this finding. If high scores on the AQ tap solely
ASD characteristics in both autistic cisgender and autistic
transgender groups, then these results indicate an increased
severity of ASD in autistic transgender people. If this is
true, autistic transgender people should also score higher
on standardized diagnostic tools for ASD (e.g., Autism
Diagnostic Observation Schedule; Lord et al., 2000) than
autistic cisgender people. To our knowledge, this has not
been examined in the literature. support and care should be provided for transgender people
regardless of whether they have a co-occurring diagnosis
of ASD (e.g., Strang et al., 2018b). Next, we examined whether autistic cisgender people
report increased current gender dysphoric feelings and
recall diminished gender-typed behavior from childhood. In
keeping with George and Stokes’ (2018b) findings, we found
that autistic cisgender individuals reported significantly
more gender dysphoric feelings than non-autistic cisgender
people, but significantly less than autistic and non-autistic
transgender people. We also extended this finding further
by showing, for the first time, that autistic cisgender people
recalled less gender-typed behavior in their childhood
memories than non-autistic cisgender individuals, but more
than autistic and non-autistic transgender people. Gender Dysphoric Feelings and Recalled
Gender‑Typed Behavior in Autism Spectrum
Disorder Nonetheless, as dis-
cussed above, Kung (2020) did not control for the presence of
autistic participants in the transgender sample. Furthermore,
we found, for the first time, that autistic transgender people
scored significantly lower on the RMIE than non-autistic
cisgender and non-autistic transgender people, indicating a
mentalizing difficulty. Noteworthy, non-autistic transgender
people performed significantly better on the task than autistic
cisgender people. Autistic‑like Traits and Mentalizing Ability
in Transgender Individuals research has shown a significant relation between mental-
izing ability and gender dysphoric feelings among people
from the general population (Kallitsounaki & Williams,
2020b; Kallitsounaki et al., 2021). Yet, the findings of
the current study indicate that this relation does not hold
among non-autistic transgender people and thus contradict
the hypothesis that mentalizing is the shared underling
mechanism that explains the high co-occurrence of ASD
and gender diversity (Glidden et al., 2016; Jacobs et al.,
2014; van der Miesen et al., 2016). Based on previous
findings, however, we cannot exclude the possibility that
mentalizing might play a role in the development of sub-
clinical gender dysphoric feelings in autistic cisgender
people (Kallitsounaki & Williams, 2020b; Kallitsounaki
et al., 2021). Of course, further research and replication of
the current findings is required before major conclusions
can be made. To increase confidence in the veracity of the
current findings, future research might usefully examine
mentalizing abilities in transgender (autistic and non-
autistic) children as well as in their parents and siblings,
employing not only explicit, but also implicit measures of
mentalizing (e.g., Senju et al., 2009). (e.g., Genderqueer Identity scale; McGuire et al., 2019) that
tap gender identification beyond binary categories. i
We should also acknowledge that in the current study we
did not collect information on co-occurring mental health
conditions. Given that co-occurring mental health conditions
are prevalent in both autistic and transgender populations
(e.g., Dhejne et al., 2016; Lai et al., 2019), and might have
an impact on some of the findings of the current study (e.g.,
increased ASD traits in non-autistic transgender individuals),
future research might address this limitation. Lastly, we should note that some of the stimuli in the
“Other” category of the IAT were in fact gender neutral
pronouns (e.g., they, them, etc.). Given that gender neutral
pronouns are commonly used by gender diverse people, it
could be argued that this methodological limitation might
have attenuated the scores of transgender participants on the
IAT. We should note, however, that in the current study we
included mostly participants who identified with a binary
gender, and thus they were more likely to use female or
male pronouns than neutral pronouns. Autistic‑like Traits and Mentalizing Ability
in Transgender Individuals Also the IAT task is
designed to be easier when two strongly associated concepts
(e.g., Self and Experienced Gender) share the same response
option than when two unrelated or only weakly related
concepts share the same response option (e.g., Self and Other
Gender; Nosek et al., 2007). On this basis, we would expect
even a transgender male who uses gender neutral pronouns to
respond faster and more accurately when words related to self
(e.g., I, me, mine) share the same response key with gender
groups that correspond to their experienced gender (e.g.,
male, son, sir, etc.) than when words related to self share the
same response key with gender groups of the opposite binary
gender (e.g., girl, female, madam, etc.). Of course, in future
studies it might be useful to collect information on pronoun
use when employing IATs that tap self-concepts. This would
provide a clear answer on whether the use of neutral pronouns
might have an effect on participant score. Limitations and Directions for Future Research Although the findings of this study enhance our understanding
of the high co-occurrence between ASD and gender diversity
by elucidating some core aspects of this phenomenon, we
should note potential limitations in this study. In order for
autistic participants to take part in the current study, they
had to report possession of a formal diagnosis of ASD. We
cannot exclude the possibility, however, that self-diagnosed
individuals also took part in the study. Future online studies
should account for the issue of sample identifiability (i.e.,
internally valid, but not necessarily representative, results;
Rubenstein & Furnie, 2021), by asking participants to provide
a copy of their diagnostic letter (see Warrier et al., 2020). Despite this potential limitation, it is important to mention
that autistic participants in this study showed clinical levels
of ASD, as well as mentalizing difficulties. The presentation
of ASD diagnosis is the autistic group of the current study
was in keeping with that found in other studies where not only
evidence of a formal diagnosis of ASD has been provided by
autistic participants, but also an independent validation of
their diagnosis has been made (e.g., Nicholson et al., 2019). F
h
i
hi
d
d
di
i
l Autistic‑like Traits and Mentalizing Ability
in Transgender Individuals From a theoretical perspective, these results add to the
discussion as to whether ASD-like traits in transgender
people represent true ASD characteristics (Fortunato et al.,
2022; Turban, 2018; Turban & van Schalkwyk, 2018). We found that non-autistic transgender people showed
increased ASD-like traits, albeit intact mentalizing abili-
ties. In contrast, autistic transgender people showed not
only increased ASD-like traits, but also potential mentaliz-
ing difficulties. Based on these results, it could be argued
that ASD-like traits in non-autistic transgender people do
not reflect “true” ASD characteristics, whereas they do tap
ASD among autistic transgender individuals. Furthermore, As predicted, non-autistic transgender individuals reported
significantly more ASD-like traits than non-autistic cisgender
individuals, but significantly fewer than autistic people (either
cisgender or transgender). This is important because to date,
only a very small number of studies (i.e., Jones et al., 2012;
Murphy et al., 2020; Warrier et al., 2020) have examined
whether increased ASD-like traits are observed in samples
of non-autistic gender diverse adults. In this study we also
found that autistic transgender people reported significantly
more ASD-like traits than autistic cisgender individuals. This
is in keeping with previous research findings (Walsh et al., 1 3 3 Archives of Sexual Behavior research has shown a significant relation between mental-
izing ability and gender dysphoric feelings among people
from the general population (Kallitsounaki & Williams,
2020b; Kallitsounaki et al., 2021). Yet, the findings of
the current study indicate that this relation does not hold
among non-autistic transgender people and thus contradict
the hypothesis that mentalizing is the shared underling
mechanism that explains the high co-occurrence of ASD
and gender diversity (Glidden et al., 2016; Jacobs et al.,
2014; van der Miesen et al., 2016). Based on previous
findings, however, we cannot exclude the possibility that
mentalizing might play a role in the development of sub-
clinical gender dysphoric feelings in autistic cisgender
people (Kallitsounaki & Williams, 2020b; Kallitsounaki
et al., 2021). Of course, further research and replication of
the current findings is required before major conclusions
can be made. To increase confidence in the veracity of the
current findings, future research might usefully examine
mentalizing abilities in transgender (autistic and non-
autistic) children as well as in their parents and siblings,
employing not only explicit, but also implicit measures of
mentalizing (e.g., Senju et al., 2009). Conclusion In sum, the main findings of this study indicate that (a) ASD
appears to hinder the consolidation of a strong explicit (and
implicit among birth-assigned females) gender self-concept
in autistic cisgender people only, (b) autistic cisgender people
have increased current gender dysphoric feelings and recall
limited gender-typed behavior from childhood, and (c) non-
autistic transgender people show only the behavioral features
of ASD, whereas autistic transgender people show features
of ASD at the behavioral and cognitive level. The current
study enhances our understanding of the link between ASD
and gender diversity. We hope future research will further
extend the evidence base required to better inform clinical
judgement and improve best practice. Furthermore, in this study we used two dimensional meas-
ures, one that measures current gender dysphoric feelings
(i.e., GIDYQ) and one that taps recalled gender-typed behav-
ior in childhood (i.e., RCGI). Although both measures are
commonly used in research, they do not capture non-binary
gender identification that is common in ASD (e.g., Walsh
et al., 2018). Future research might usefully employ measures 1 3 Archives of Sexual Behavior Supplementary Information The online version contains supplemen-
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Imported loiasis in France: a retrospective analysis of 167 cases with comparison between sub-Saharan and non sub-Saharan African patients
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To cite this version: Olivier Bouchaud, Sophie Matheron, Anne Loarec, Jean Dupouy Camet, Patrice Bourée, et al.. Im-
ported loiasis in France: a retrospective analysis of 167 cases with comparison between sub-Saharan
and non sub-Saharan African patients. BMC Infectious Diseases, 2020, 20 (1), pp.63. 10.1186/s12879-
019-4740-6. hal-02499338 Imported loiasis in France: a retrospective analysis of
167 cases with comparison between sub-Saharan and
non sub-Saharan African patients Olivier Bouchaud, Sophie Matheron, Anne Loarec, Jean Dupouy Camet,
Patrice Bourée, Nadine Godineau, Isabelle Poilane, Johann Cailhol, Eric
Caumes HAL Id: hal-02499338
https://hal.sorbonne-universite.fr/hal-02499338v1
Submitted on 5 Mar 2020 L’archive ouverte pluridisciplinaire HAL, est
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abroad, or from public or private research centers. Bouchaud et al. BMC Infectious Diseases (2020) 20:63
https://doi.org/10.1186/s12879-019-4740-6 Bouchaud et al. BMC Infectious Diseases
https://doi.org/10.1186/s12879-019-4740-6 (2020) 20:63 Open Access Imported loiasis in France: a retrospective
analysis of 167 cases with comparison
between sub-Saharan and non sub-Saharan
African patients Olivier Bouchaud1*
, Sophie Matheron2, Anne Loarec1, Jean Dupouy Camet3, Patrice Bourée4, Nadine Godineau5,
Isabelle Poilane6, Johann Cailhol1 and Eric Caumes7 Abstract Background: Imported loiasis is a rare cause of consultation at the return of stay in central Africa, which often
poses difficult diagnostic and therapeutic questions to practitioners especially those who are unaccustomed to
tropical medicine. These difficulties can lead to risks for the patients especially if inappropriate treatment is given. Large series of imported loiasis are scarce. Methods: We retrospectively studied the data including outcome in patients diagnosed with imported loiasis
between 1993 and 2013 in the Paris area on the basis of a parasitological diagnosis (microfilaremia > 1/ml and/or
serologic tests). We compared sub-Saharan and non sub-Saharan African patients. Results: Of the 177 identified cases, 167 could be analysed. Sex ratio was 1, mean age 41 years and 83% were
sub-Saharan Africans. Cameroon was the main country of exposure (62%). Incubation time may be long (up to 18
months). Of the 167 cases, 57% presented with characteristic symptoms (Calabar swellings, creeping dermatitis,
eyeworm) whereas 43% were diagnosed fortuitously. Microfilaremia was evidenced in 105 patients (63%), and
specific antibodies in 53%. Compared to sub-Saharan Africans, other patients were presenting less frequently with
eyeworm migration and microfilaremia whereas they had higher eosinophilia and positive serology. Prevalence of
Calabar swellings was not significantly different between the two groups. Cure rates were 52% with ivermectin
alone, and 77% with ivermectin followed by diethylcarbamazine. No severe adverse event was reported. Conclusions: Presentation of imported loiasis varies according to ethnicity. A systematic screening should be
recommended in patients with potential exposure in endemic country. Treatment with ivermectin followed by
diethylcarbamazine could be a valuable option. : West and Central Africa, Diethylcarbamazine, Ivermectin, Loiasis, Microfilaremia, Traveller, Travel Keywords: West and Central Africa, Diethylcarbamazine, Ivermectin, Loiasis, Microfilaremia, Traveller, Travel
medicine Keywords: West and Central Africa, Diethylcarbamazine, Ivermectin, Loiasis, Microfilaremia, Traveller, Travel
medicine * Correspondence: olivier.bouchaud@aphp.fr * Correspondence: olivier.bouchaud@aphp.fr
1Infectious Diseases and Tropical Medicine Department, Avicenne Hospital
and Paris 13 University, 93000 Bobigny, France
Full list of author information is available at the end of the article * Correspondence: olivier.bouchaud@aphp.fr
1Infectious Diseases and Tropical Medicine Department, Avicenne Hospital
and Paris 13 University, 93000 Bobigny, France
Full list of author information is available at the end of the article Background lasting (less than 1 week) painless oedema of the extrem-
ities (joints, legs, arms or face). Other forms of subcuta-
neous
oedema
with
a
different
location
or
more
prolonged duration were distinguished from Calabar
swelling. Eye or subcutaneous worm migration was de-
fined by the history of a temporary creeping lesion under
the conjunctiva or the skin, leaving no trace behind, no-
ticed by the patient and/or the physician. Ocular symp-
toms other than eye worm migration were analyzed
separately. g
Loiasis, caused by Loa loa and transmitted by bites of
tabanid flies of the genus chrysops is endemic in the
forested areas of Western and Central Africa [1–4]. Loiasis is rarely diagnosed in returning travellers being
found in only 68 of 43,722 ill returning travelers
(0.17%) [5]. Nine series of imported loiasis (IL) have
been published over the last 30 years [6–14]. Most of
them included a limited number of cases. The three
largest studies including 100 cases for two of them
and 186 for the third one, took place in England, Italy
and the United States, respectively. In these three
studies, characteristics of disease were compared be-
tween Africans and expatriates [8, 11, 13]. Diagnosis
of loiasis is often difficult, and complications may be
precipitated by inappropriate treatment. Indeed, in
case of high microfilaremia, treatment with diethylcar-
bamazine (DEC) or ivermectin may lead to systemic
inflammatory reactions including life-threatening enceph-
alitis classically assigned to parasite lysis [1–3, 6, 15]. p
y
Hypereosinophilia was defined by an absolute blood
eosinophilic count > 500/mm3. Microfilaremia was de-
termined by the microscopic observation of Loa loa
microfilariae in a blood smear (firstly on a drop of fresh
blood and secondly, when microfilariae were visualized,
on a thick film after staining for confirmation and
counting). In the case of negative microscopic examin-
ation, the search for microfilariae was considered nega-
tive after leucoconcentration over five milliliters was also
negative. Different techniques of serology were used,
each parasitology department having their own, but all
considering at least two techniques for concluding to
positivity including one or two screening tests and, in
case of positivity, one or two confirmation techniques. Thus serology was considered positive if at least 2 tests
were positive, the first being a screening and the second 1 a
confirmation test. According to this rule, the different com-
binations of screening and confirmation techniques were as
follows: i. Background immunofluorescence using Molinema dessetae
antigens and/or ELISA with Toxocara canis antigens
confirmed by an ouchterlony technique with an immuno-
diffusion using Ascaris suum antigens and/or an im-
munoelectrophoresis method with Loa loa specific anti-
gen; ii. direct or indirect immunofluorescence and/or
ELISA confirmed by counter-electrophoresis and/or
immunoelectrophoresis with Ascaris suum antigens; iii. Immunofluorescence using Molinema dessetae antigens
confirmed by co-electrosyneresis using Ascaris suum
antigens. Apart from the ELISA tests, which were com-
mercial kits, these techniques were home-made and the
threshold of positivity was defined by each laboratory. In case of serology classified as “undetermined” by the
laboratory when it was not negative but under the
threshold of positivity for each technique, we classified
the result as negative We report 167 cases observed within a 20 years-period
in the Paris area with a particular attention to the differ-
ences between sub-Saharan Africans and other patients. © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bouchaud et al. BMC Infectious Diseases (2020) 20:63 Bouchaud et al. BMC Infectious Diseases (2020) 20:63 Page 2 of 7 Methods We retrospectively analyzed the epidemiological, clinical,
and biological data as well as treatment and outcome of
all the patients diagnosed with IL between January 1993
and December 2013 in nine hospitals in Paris and its
suburbs. These hospitals were selected because they are
located in areas with a high density of African immi-
grants or they have a clinical or parasitological depart-
ment involved in tropical medicine. All the patients with a parasitological diagnosis of lo-
iasis including positive microfilaremia (> 1/ml) and/or
positive serologic tests were selected. Then, for patients
diagnosed serologically, considering the limitations of
serological tests, only patients with an epidemiological
(stay in endemic areas) and/or a clinical presentation
compatible with a loiasis were definitively included. Two
populations of patients were distinguished. Sub-Saharan
African (SSA) patients were defined as immigrants (born
in endemic areas of sub-Saharan Africa, living in France)
with a history of travel to their country of origin for vis-
iting friends and relatives (VFR), and those living in en-
demic areas of sub-Saharan Africa visiting/arriving in
France for various purposes. In SSA-VFR patients, we
considered the last travel as that at risk of exposure to
loiasis. Non sub-Saharan African (non-SSA) patients
were defined as patients originating from Europe or
North-Africa with a history of travel to endemic coun-
tries for loiasis. The country of acquisition was deter-
mined according to the patient’s travel characteristics. Calabar swelling was defined as recurrent and short- We assessed the epidemiological data (age, sex, ethnicity,
country of origin, last visited country before diagnosis, char-
acteristics of travel), clinical aspects (medical history, reason
of first consultation, description and duration of symptoms)
and biological results (blood cells count, creatininemia,
transaminases, filariasis serology and microfilaremia count)
in patients with IL. We compared these data in SSA versus
non-SSA
patients,
and
in
symptomatic
versus
non-
symptomatic patients. We also evaluated the sensitivity of Bouchaud et al. Methods BMC Infectious Diseases (2020) 20:63 Page 3 of 7 Table 1 Epidemiological and clinical characteristics in 167
patients with imported loiasis
N
%
Age
[0–16]
9
5.3
[16–59]
131
78.4
[60-]
27
16.1
Sex
Female
83
49,7
Male
84
50.2
Ethnic group
Sub-Saharan Africans
139
83,2
Non sub-Saharan Africansa
28
16.7
Country of acquisition
Cameroon
104
62.2
Gabon
27
16.1
Congo-Brazzaville
16
9.5
Central African Republic
6
3.5
Othersb
8
4.7
Undetermined
6
3.8
Symptoms c,d
Itching
74
44.3
Calabar swelling
54
32,3
Subcutaneous oedema
29
17.3
Eyeworm
39
23.3
Other ocular symptoms
24
14.3
Subcutaneous worm migration
8
4.7
No symptom
45
26.9
a
b
h
f
(
)
d
f Table 1 Epidemiological and clinical characteristics in 167
patients with imported loiasis serology compared to that of direct diagnosis by microfilar-
emia detection for diagnosing IL. The treatment, depending of each physician’s choice, al-
ways included ivermectin and/or DEC. DEC was given at
a progressively increasing daily dosage, up to 400 mg per
day, with a duration of 21 days once the dosage arrived at
full dose. Full cure was defined as the absence of clinical
symptoms and negative microfilaremia at the latest
follow-up visit. “Partial” cure was defined as disappearance
of clinical symptoms and decreased microfilaremia. Failure
was stated when signs and symptoms persisted and micro-
filarial levels did not change significantly. Post-treatment
reaction was defined by the occurrence of symptoms
(fever, pruritus, angio-oedema, malaise, hypotension, fa-
tigue, vertigo, headaches, joint or muscle or abdominal
pain, central nervous system manifestations) within the
24 h following ivermectin or DEC administration. We used Chi2 of Pearson and Mann-Whitney test (non
parametric test for continuous variables) for statistical tests
with Epi Info™software (version 7.2, 2016, Atlanta, USA). As patient data were initially collected as part of rou-
tine care and no additional examination was performed,
agreement of an ethics committee was not required ac-
cording to the French regulation at the time of the start
of the study but the database was declared to the CNIL
(Commission Nationale Informatique et Liberté). All
data were completely anonymized in each of the centres
that participated in the study. Methods aNon sub-Saharan African patients = Europeans (N = 26) and patients from
North Africa (N = 2)
bother countries: Benin, Ivory Coast, Equatorial Guinea, Mali, Rwanda,
Democratic Republic of Congo
ctotal percentage of different symptoms exceeds 100% as one patient may
have presented several symptoms simultaneously
dmain data from the 3 patients who reported non-endemic countries as
countries of contamination (see discussion): Ivory Coast: VFR, itching,
microfilaremia: 3/mL; Mali (South): VFR, stay of 3 months, itching,
microfilaremia: 4/mL; Rwanda: VFR, subcutaneous oedema (ankle), serology +
with specific arc at immunoelectrophoresis Results Among the 177 identified cases of IL, analyzable data were
available in 167, included in the present study. Sex ratio was
1.01 (84 men and 83 women), and mean age was 41.2 years
(Table 1). SSA patients accounted for 83.2%. Cameroon was
the leading country of exposure (62.2%), followed by Gabon
and Congo-Brazzaville. Sex ratio was 0.88 among SSA pa-
tients, and 2.3 in non-SSA patients. In SSA-VFR patients
and non-SSA patients, in whom the data may be calculated,
the median duration of the at-risk travel was 3 months (IQR
3–30). The time between return to France and onset of
symptoms could be evaluated in nine patients because they
travelled in the at-risk country only once, and was estimated
at 12 months (range: 6–18 months). patients experienced subcutaneous oedema. Eyeworm
was described in 39 patients. Ocular symptoms other
than sub-conjonctival crossing were pain (N = 6), con-
junctivitis (N = 4), eyelid oedema (N = 5), ocular discom-
fort (N = 4), and other symptoms for 5 patients. One hundred and two patients (61%) had microfilaremia
with a mean value of 1822/ml (range: 1–50,000/ml)
whereas 53 (31.7%) had a negative microfilaremia; data
were missing in 12 cases. Ninety-two patients (55%) had a
positive serology, including 54 with a specific arc evi-
denced by immunoelectrophoresis (performed in 125 pa-
tients). For patients for whom serology and microfilaremia
results are available, 24 patients had positive microfilar-
emia and negative serology, 44 had negative microfilar-
emia and positive serology and 46 had microfilaremia
and positive serology. Spontaneous reporting of symptoms of loiasis moti-
vated the initial consultation in 95 patients (56.8%). In
43.2% the diagnosis was considered because of hypereo-
sinophilia or during systematic examination after return
from endemic country evidencing suggestive symptoms
(mainly pruritus) that led to diagnose loiasis by micros-
copy and serology. Overall 122 (73.1%) of the patients
were symptomatic. Itching was present in 74 patients
(44.3%). A history of creeping dermatitis was found in 8
patients. Calabar oedema were observed in 54 patients,
mostly on wrists (N = 31) or legs (N = 23), and 29 Clinical and biological features were compared be-
tween SSA and non-SSA patients (Table 2). Compared
to SSA patients, non-SSA patients were more likely to Bouchaud et al. Results BMC Infectious Diseases (2020) 20:63 Page 4 of 7 Table 2 Comparison between sub-Saharan African (SSA) and non sub-Saharan (non-SSA) African patients with imported loiasis
SSA patients
N = 139
non-SSA patients
N = 28
p
N
%
N
%
Prior history of loiasis
34
24.4
14
50
0.01
Asymptomatic for loiasis
39
28.5
6
21.4
NS
Calabar swelling
43
30.9
11
39.2
NS
Eyeworm
36
25.8
3
10.7
0.05
Mean eosinophilia (/mm3)
1591
2854
0.04
Microfilaremia
positive
94
67.6
11
39.2
0.05
negative
39
28
16
57.1
missing
7
0
Meana microfilaremia (/ml)
2586
1247
NS
Serology
positive
64
46
24
85.7
0.04
negative
30
21.5
2
7.1
undetermined
16
0
missing
30
1
NS not significant; aarithmetic means calculated on microfilaraemic subjects Table 2 Comparison between sub-Saharan African (SSA) and non sub-Saharan (non-SSA) African patients with imported loiasis Outcome was evaluable in 165/167 patients including
149 treated patients and 16 patients who did not receive
any treatment (loss of follow-up, pregnancy, frequent
travels planned in their country of origin) (Table 4). Most patients received ivermectin alone (75.8%) or
followed by DEC (17.4%) whereas 10 patients (6.7%)
received DEC only. Ivermectin was given either as a sin-
gle course (7.1%) or repeated courses (92.9%). A pre-
ventive
treatment
of
post-treatment
reaction
(anti-
histaminic and/or corticosteroids) was given in 26 pa-
tients. Mean time of follow-up was 6 months (range: 1–34
months). Full cure rate was 52.2% in the patients treated
with ivermectin alone (1 to 6 courses), and 76.9% in those
who received ivermectin followed by one course of DEC. Eleven patients (7.3%) experienced post-treatment reac-
tion (4.4% following ivermectin alone, 20% following DEC
alone, 15.3% following ivermectin + DEC), consisting in have a prior history of loiasis (p = 0.01), a higher blood eo-
sinophilia count (p = 0.04), and to have a positive serology
(p = 0.04). SSA patients were more likely to have microfi-
laremia than non-SSA patients (p = 0.05), but their mean
microfilaremia did not differ significantly (p = 0.42). microfilaremia did not differ significantly (p = 0.42). Comparing biological results in asymptomatic and
symptomatic patients, no difference was observed in mean
eosinophilia, rates of positive microfilaremia and positive
serology, and the mean number of parasites/ml (Table 3). Diagnosis sensitivity of serology was assessed in com-
parison to microfilaremia detection (data not shown). Among patients with a definite diagnosis (i.e. Discussion This large study of imported loiasis shows that loiasis
may be asymptomatic, may appear long time after return
(up to 18 months) and that the hypothesis of persistent
low transmission in formerly endemic areas could be
further investigated. It also contributes to assess the
response to treatment although the limitations of a
retrospective study should lead to caution in interpreting
these results. A high proportion (43.2%) of our patients was fortuit-
ously diagnosed because history of compatible but mild
or non specific symptoms and/or hyperereosinophilia
after returning from endemic countries. This point has
not been highlighted in other series of IL because
patients were generally included only on the basis of
specific symptoms [1–3]. As a result we recommend that
every patient at risk of loiasis (ie: having lived or trav-
elled for a long time in endemic areas even long time
ago) should be systematically screened for loiasis. In our study the leading country of acquisition of IL
was Cameroon, in agreement with the results of two
other French studies [6, 10]. In England, the leading
country of acquisition is Nigeria [8]. This is not surpris-
ing as loiasis is highly endemic in Cameroon and
Nigeria, and as both countries account for a high num-
ber of migrants in France, and England respectively, ac-
cording to colonial history. Overall 73% of our patients were symptomatic, with
classic symptoms (itching, Calabar oedema, creeping
dermatitis, eye worm) but also with less characteristic
clinical manifestations. Itching was a complaint in nearly
two thirds of our symptomatic population, compared to
one third of that reported by Churchill et al. [8] Calabar
oedema and migratory oedema were reported in respect-
ively 44 and 24% of our symptomatic patients, whereas
Calabar swellings were reported in, respectively, 62 and
74.7% of symptomatic cases by Churchill and Herrick [8,
13]. However, we differentiated sensu stricto Calabar
oedema from migratory oedemas which can explain such
difference [17]. We also differentiated ocular symptoms However four patients were found to have been in-
fected in countries where loiasis is not currently consid-
ered to be endemic (Mali, Benin, Ivory Coast, Rwanda)
even if a limited focus of loiasis has been described in
the south-east part of Benin [1, 2, 16]. This has not been
showed in other studies of IL. Results BMC Infectious Diseases (2020) 20:63 Page 5 of 7 Table 4 Treatment outcomes in 149 patients with imported loiasis Table 4 Treatment outcomes in 149 patients with imported loiasis
ivermectinab
diethylcarbamazinecd
ivermectina then diethylcarbamazinec
N = 113 (75.8%)
N = 10 (6.7%)
N = 26 (17.4%)
N
%
N
%
N
%
Outcomes:
failure
10
8.8
2
20
2
7.6
full cure
59
52.2
0
0
20
76.9
partial cure
0
0
5
50
2
7.6
loss of follow up
44
38.9
3
30
2
7.6
preventive treatment of post-treatment reaction
15
13.2
4
40
7
26.9
post-treatment reaction
5
4.4
2
20
4
15.3
abetween 1and 6 courses (1 course: 62.8%; 2: 17.8%; 3: 7.7%; 4: 6.2%; 5: 2.3%; 6: 3.1%), 10 (7.1%) patients received only 1 course, associated with albendazole in
4 cases
b200 μg/kg per course
cprogressive dosage (initial dosage between 10 and 75 mg, final dosage 200–400 mg for 21 days)
dTwo patients with a high microfilaremia (38,200 and 50,000/mL, respectively) were treated with filariopheresis followed by diethylcarbamazine without significant
adverse event 200 μg/kg per course
cprogressive dosage (initial dosage between 10 and 75 mg, final dosage 200–400 mg for 21 days)
dTwo patients with a high microfilaremia (38,200 and 50,000/mL, respectively) were treated with filariopheresis followed by diethylcarbamazine without significant
d
t fever, malaise, fatigue, headache, and/or abdominal pain. No severe adverse event (including encephalopathy) was
reported. No post-treatment reactions were reported in
patients who received preventive treatment with anti-
histaminic and/or corticosteroids. Studies of IL usually fail to determine incubation
period since it is not possible to estimate the date of ac-
quisition neither in patients native from endemic coun-
try nor in long term travellers or travellers traveling
frequently to endemic areas. We estimated the median
incubation time at 12 months in the nine patients with
data allowing this evaluation. If this median incubation
time cannot be extrapolated to all patients in the study,
it is important to note for the clinician that clinical signs
of loiasis may appear long after return and up to 18
months in our study. This duration is in agreement with
that found in the only other study in which this param-
eter was evaluable [6]. fever, malaise, fatigue, headache, and/or abdominal pain. No severe adverse event (including encephalopathy) was
reported. No post-treatment reactions were reported in
patients who received preventive treatment with anti-
histaminic and/or corticosteroids. Results proven by
positive microfilaremia) and also with positive serology,
the serology sensitivity was estimated at 69%. Sensitivity
of serology among patients without microfilaremia but
presenting clinical symptoms concordant with loiasis
was estimated at 96%. Comparing biological results in asymptomatic and
symptomatic patients, no difference was observed in mean
eosinophilia, rates of positive microfilaremia and positive
serology, and the mean number of parasites/ml (Table 3). Diagnosis sensitivity of serology was assessed in com-
parison to microfilaremia detection (data not shown). Among patients with a definite diagnosis (i.e. proven by
positive microfilaremia) and also with positive serology,
the serology sensitivity was estimated at 69%. Sensitivity
of serology among patients without microfilaremia but
presenting clinical symptoms concordant with loiasis
was estimated at 96%. Table 3 Comparison between asymptomatic and symptomatic patients with imported loiasis
Asymptomatic patients N = 45
Symptomatic patients N = 122
p
N
%
N
%
Mean eosinophilia (/mm3)
1902
2026
NS
Microfilaremia
positive
34
75.5
68
55.7
NS
negative
9
20
44
36
missing
2
10
Mean microfilaremia (/ml)
1092
2101
NS
Serology
positive
23
51.1
69
56.50
NS
negative
9
20
17
13.9
undetermined
3
13
missing
10
23
NS not significant Table 3 Comparison between asymptomatic and symptomatic patients with imported loiasis
Asymptomatic patients N = 45
Symptomatic patien Bouchaud et al. Discussion According to some au-
thors, the western part of the African rain forest has
been considered in the past as a possible endemic zone
for loiasis [2, 16]. Although it is most likely that these
patients have forgotten to mention a stay in an en-
demic area, it is possible to hypothesize persistent
transmission at a low level in isolated areas of these
formerly endemic areas. Page 6 of 7 Page 6 of 7 Bouchaud et al. BMC Infectious Diseases (2020) 20:63 Bouchaud et al. BMC Infectious Diseases (2020) 20:63 endemic countries, at least one course of ivermectin
followed by one course of DEC appears to be a good
option to reach an acceptable cure rate without taking
the risk of severe adverse event. This is consistent with a
93% reduction of microfilaremia observed in seven pa-
tients treated by ivermectin before receiving DEC. [27]
This option seems particularly adequate when microfi-
larial density is relatively high (between 2000 and 8000/
ml) while a density below 2000/ml allows to initiate the
cure directly with DEC according to Boussinesq [28]. Adverse events following DEC (and at a lesser extent
ivermectin) have been reported both in endemic zones
and in IL [1–3, 6, 8, 15]. The higher is the parasite load
the higher is the risk of developing marked or serious
adverse events such as encephalopathy when microfilar-
emia is above 50,000/ml [9, 12]. Recent data suggest
that post-treatment reactions following DEC and iver-
mectin occur earlier with DEC but share a common
pathophysiology [29]. from eye worm migration because one of our patients pre-
sented an intra-vitreous haemorrhage, a complication
rarely reported [3, 18, 19]. Creeping dermatitis was found
in about 5% of our patients but loiasis is a very rare cause
of creeping dermatitis, been found in only one amongst 70
patients consulting for creeping dermatitis [20]. p
g
p g
We found some significant differences related to ethni-
city as non-SSA patients were found with less frequent
eyeworm, higher eosinophilia, fewer detectable microfilar-
emia, and more common positive serology compared to
SSA patients. We thus confirm the results found in five
other comparative studies [8, 10–13]. Such differences
have been previously described and were attributed to a
possible immune tolerance in Africans with multiple
exposures to the parasite [2, 7, 8, 10–13, 21–24]. Discussion Some au-
thors highlighted the major role played by the antibodies-
mediated immune response (with notably specific IgG
antibodies) in cooperation with cellular immunity includ-
ing lymphocyte proliferation to parasites antigens [7, 12,
16, 25]. In keeping with this hypothesis, we found that the
sensitivity of serology was higher among patients without
detectable microfilaremia, suggesting an immune mechan-
ism which controls the multiplication of parasites. Similar
results were found by Churchill with a better sensitivity of
serology in expatriates compared to Africans [8]. Herrick
hypothesises that differing eosinophil-associated responses
to the parasite may be responsible for the differences in
clinical presentations [13]. Our study has some limitations that mostly concern
inclusion criteria and treatment mainly due to the retro-
spective design of the study. The first limitation is the
lack of standardization of diagnostic tests because, if the
criterion of positivity was the same for all patients (at
least 2 positive serological tests including a screening
test and a confirmation test), the techniques used and
the positivity thresholds varied from one centre to an-
other. However, since for patients diagnosed by serology
alone we only considered those with an epidemiological
and clinical history compatible with a loiasis diagnosis
and excluded cases with serologies for which the result
was not clearly positive, we believe that the risk of mis-
diagnosis is limited. Another limitation is the heterogen-
eity of treatment regimens, the number of patients lost
to follow up, and the limited duration of treatment fol-
low up, but these limitations are shared by other studies
that had fewer patients than ours. Current diagnostic tools have limitations and more ef-
fective tests are needed in both endemic areas or in the
frame of IL. Recently, a rapid antibody-detection test has
been developed [26]. Should the first encouraging results
be confirmed, this new tool would certainly be most use-
ful to diagnose loiasis, especially in its occult (amicrofi-
laraemic) forms. One or more courses of ivermectin, alone and followed
by one course of DEC, gave a cure rate of 52 and 77%,
respectively, with a low rate of adverse events and no se-
vere adverse event. Similar results have been found in
smaller studies. Churchill et al. showed a cure rate of
63% (with no difference between Africans and non-
Africans) and a relapse rate of 12% among 100 patients
who were mainly treated with diethylcarbamazine [8]. Klion et al. Abbreviations Abbreviations
DEC: Diethylcarbamazine; IL: Imported loiasis; IQR: Interquartile; SSA: Sub-
Saharan African patients; VFR: Visiting friends and relatives DEC is the corner-stone of the treatment of loiasis due
to its macrofilaricidial activity (in contrast to ivermectin
or albendazole). Therefore, in patients living in non- Discussion reported, in 32 expatriates followed up dur-
ing a median time of 4.5 years, a cure rate of 38% after
one course of DEC, and 16% after two courses, whereas
53% relapsed, within the first year for the majority [7]. El
Aouri reported eight relapses (31%) among 26 expatri-
ates returning from Equatorial Guinea and treated with
DEC (N = 15), ivermectin plus DEC (N = 9) or ivermectin
alone (N = 2) [9]. Conclusions We recommend to systematically consider loiasis in all
patients returning from endemic countries with either
hypereosinophilia,
pruritus
or
recurrent
oedema
in
addition to the more classic signs, even several months
or years after return. The association of one or more
courses of ivermectin followed by at least one course of
DEC appears a valuable option for treating imported
loiasis and needs to be evaluated as well as the use of
albendazole which has not been assessed in this setting. Abbreviations
DEC: Diethylcarbamazine; IL: Imported loiasis; IQR: Interquartile; SSA: Sub-
Saharan African patients; VFR: Visiting friends and relatives Funding
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after mass treatment of onchocerciasis with ivermectin in an area endemic
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to collect the data analyzed in this work. The authors thank all the physicians and the parasitologists who contributed
to collect the data analyzed in this work. Page 7 of 7 Bouchaud et al. BMC Infectious Diseases (2020) 20:63 Consent for publication
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published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
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Millennial-age GDGTs in forested mineral soils: &lt;sup&gt;14&lt;/sup&gt;C-based evidence for stabilization of microbial necromass
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ETH Library Publication date:
2020-08-26 Rights / license:
Creative Commons Attribution 4.0 International g
Creative Commons Attribution 4.0 International Author(s): Author(s):
Gies, Hannah; Hagedorn, Frank; Lupker, Maarten; Montluçon, Daniel; Haghipour, Negar; van der Voort, Tessa S.; Eglinton,
Timothy I. ( )
Gies, Hannah; Hagedorn, Frank; Lupker, Maarten; Montluçon, Daniel; Haghipour, Negar; van der Voort, Tessa S.; Eglinton,
Timothy I. Millennial-age GDGTs in forested mineral soils: 14C-based evidence
for stabilization of microbial necromass
Hannah Gies1, Frank Hagedorn2, Maarten Lupker1, Daniel Montluçon1, Negar Haghipour1,3, Tessa
Sophia van der Voort4 and Timothy Ian Eglinton1 1Department of Earth Sciences, ETH Zürich, Sonneggstrasse 5, 8092 Zürich, Switzerland
2Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Zürcherstrasse 111, 8903 Birmensdorf, Switzerland
3Laboratory of Ion Beam Physics, ETH Zürich, Otto-Stern-Weg 5, 8093 Zürich, Switzerland
4Campus Fryslan, Rijksuniversiteit Groningen, Wirdumerdijk 34, 8911 CE Leeuwarden, Netherlands
Correspondence: Hannah Gies (hannah gies@erdw ethz ch) Abstract. Understanding controls on the persistence of soil organic matter (SOM) is essential to constrain its role in the carbon
cycle and inform climate-carbon cycle model predictions. Emerging concepts regarding formation and turnover of SOM imply
that it is mainly comprised of mineral-stabilized microbial products and residues, however, direct evidence in support of this
concept remains limited. Here, we introduce and test a method for isolation of isoprenoid and branched glycerol dialkyl glycerol tetraethers (GDGTs) – diagnostic membrane lipids of archaea and bacteria, respectively - for subsequent natural abundance
5
radiocarbon analysis. The method is applied to depth profiles from two Swiss pre-alpine forested soils. We find that the ∆14C
values of these microbial markers markedly decrease with increasing soil depth, indicating turnover times of millennia in
mineral subsoils. The contrasting metabolisms of the GDGT-producing microorganisms indicates it is unlikely that the low
∆14C values of these membrane lipids reflect heterotrophic acquisition of 14C-depleted carbon. We therefore attribute the tetraethers (GDGTs) – diagnostic membrane lipids of archaea and bacteria, respectively - for subsequent natural abundance
5
radiocarbon analysis. The method is applied to depth profiles from two Swiss pre-alpine forested soils. We find that the ∆14C
values of these microbial markers markedly decrease with increasing soil depth, indicating turnover times of millennia in
mineral subsoils. The contrasting metabolisms of the GDGT-producing microorganisms indicates it is unlikely that the low
∆14C values of these membrane lipids reflect heterotrophic acquisition of 14C-depleted carbon. We therefore attribute the 14C-depleted signatures of GDGTs to their physical protection through association with mineral surfaces. These findings thus
10
provide strong evidence for the presence of stabilized microbial necromass in forested mineral soils. Originally published in: Originally published in:
Biogeosciences Discussions, https://doi.org/10.5194/bg-2020-308 g
y p
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Introduction Here we examine the 14C characteristics of Glycerol Dialkyl Glycerol Tetraethers (GDGTs) – characteristic membrane lipids
of microorganisms that are ubiquitous in terrestrial and aqueous environments (Schouten et al., 2013). GDGTs are subdivided into two groups of compounds: isoprenoid GDGTs (isoGDGTs) produced by Archaea (De Rosa and Gambacorta, 1988) and
35
branched GDGTs (brGDGTs) which are of putative bacterial origin (Weijers et al., 2006a) and are especially abundant in soils
and peats (Weijers et al., 2006b)(For molecular structures see Figure A1). GDGTs have garnered much attention due to their
potential as molecular proxies for environmental conditions: the relative abundance of branched versus isoprenoid GDGTs
has been used to qualitatively estimate soil-derived carbon input into marine sediments (Hopmans et al., 2004), while the into two groups of compounds: isoprenoid GDGTs (isoGDGTs) produced by Archaea (De Rosa and Gambacorta, 1988) and
35
branched GDGTs (brGDGTs) which are of putative bacterial origin (Weijers et al., 2006a) and are especially abundant in soils
and peats (Weijers et al., 2006b)(For molecular structures see Figure A1). GDGTs have garnered much attention due to their
potential as molecular proxies for environmental conditions: the relative abundance of branched versus isoprenoid GDGTs
has been used to qualitatively estimate soil-derived carbon input into marine sediments (Hopmans et al., 2004), while the internal distribution of iso- and branched GDGT isomers carries information of aquatic and soil conditions (Schouten et al.,
40
2002; Powers et al., 2004, 2010; Liu et al., 2013; Coffinet et al., 2014; Yang et al., 2016). For example, the distribution of
different brGDGTs, parameterized as the methylation of branched tetraethers (MBT) and cyclisation of branched tetraethers
(CBT) indices (Peterse et al., 2012; De Jonge et al., 2014; Naafs et al., 2017), have been found to correlate with mean annual
continental air temperature (MAT) and soil pH Weijers et al. (2007), respectively. internal distribution of iso- and branched GDGT isomers carries information of aquatic and soil conditions (Schouten et al.,
40
2002; Powers et al., 2004, 2010; Liu et al., 2013; Coffinet et al., 2014; Yang et al., 2016). For example, the distribution of
different brGDGTs, parameterized as the methylation of branched tetraethers (MBT) and cyclisation of branched tetraethers
(CBT) indices (Peterse et al., 2012; De Jonge et al., 2014; Naafs et al., 2017), have been found to correlate with mean annual
continental air temperature (MAT) and soil pH Weijers et al. (2007), respectively. 1
Introduction Soil organic matter (SOM) represents the largest reservoir of carbon in terrestrial ecosystems, exchanging large quantities of
carbon with the atmosphere and supplying aquatic systems with organic and inorganic C (Parry et al., 2007; Battin et al., 2009;
15
Bradford et al., 2016). SOM is comprised of a complex mixture of components that turn over on a wide range of timescales
(from seconds to millenia), introducing large uncertainties in climate model predictions (Carvalhais et al., 2014; He and Yu,
2016). Emerging concepts of SOM suggest that only a small fraction of annual C inputs from plants persists in the soils, and
that microbial products and residues stabilized by the interaction with reactive minerals comprise the majority of the soil C
pool (Schmidt et al., 2011; Cotrufo et al., 2015; Lehmann and Kleber, 2015; Kallenbach et al., 2016; Kästner and Miltner,
20 Soil organic matter (SOM) represents the largest reservoir of carbon in terrestrial ecosystems, exchanging large quantities of
carbon with the atmosphere and supplying aquatic systems with organic and inorganic C (Parry et al., 2007; Battin et al., 2009;
15
Bradford et al., 2016). SOM is comprised of a complex mixture of components that turn over on a wide range of timescales
(from seconds to millenia), introducing large uncertainties in climate model predictions (Carvalhais et al., 2014; He and Yu,
2016). Emerging concepts of SOM suggest that only a small fraction of annual C inputs from plants persists in the soils, and
that microbial products and residues stabilized by the interaction with reactive minerals comprise the majority of the soil C pool (Schmidt et al., 2011; Cotrufo et al., 2015; Lehmann and Kleber, 2015; Kallenbach et al., 2016; Kästner and Miltner,
20 1 1 https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. 2018). Correspondingly, microbial processes are increasingly being incorporated into soil carbon cycle models (Riley et al.,
2014; Ahrens et al., 2015). However, evidence of the ‘entombment’ of microbial necromass is presently limited and largely
circumstantial, being primarily based on the finding of increasing contributions of microbial biomarker as compared plant-
derived compounds with increasing soil depth (Amelung et al., 2008; Miltner et al., 2012; Kallenbach et al., 2016; Liang et al.,
2019; Ma et al 2018)
5 2018). 1
Introduction Correspondingly, microbial processes are increasingly being incorporated into soil carbon cycle models (Riley et al.,
2014; Ahrens et al., 2015). However, evidence of the ‘entombment’ of microbial necromass is presently limited and largely
circumstantial, being primarily based on the finding of increasing contributions of microbial biomarker as compared plant-
derived compounds with increasing soil depth (Amelung et al., 2008; Miltner et al., 2012; Kallenbach et al., 2016; Liang et al., 2019; Ma et al., 2018). 25
Radiocarbon provides valuable constraints on carbon turnover in soils (Trumbore et al., 1996; Schrumpf and Kaiser, 2015),
and 14C measurements are particularly useful when applied at the level of specific compounds (e.g., Van der Voort et al., 2017). Prior radiocarbon analyses of plant-derived biomarkers have indicated their stabilization in mineral soils (Huang et al., 1999;
van der Voort et al., 2016), but 14C-based evidence for stabilization of microbial necromass in SOM is currently lacking. ∆14C 2019; Ma et al., 2018). 25
Radiocarbon provides valuable constraints on carbon turnover in soils (Trumbore et al., 1996; Schrumpf and Kaiser, 2015),
and 14C measurements are particularly useful when applied at the level of specific compounds (e.g., Van der Voort et al., 2017). Prior radiocarbon analyses of plant-derived biomarkers have indicated their stabilization in mineral soils (Huang et al., 1999;
van der Voort et al., 2016), but 14C-based evidence for stabilization of microbial necromass in SOM is currently lacking. ∆14C 25 signatures of fatty acids and phospholipid-fatty acids (PLFAs), established indicators for plant and microbial-derived C, suggest
30
active microbial re-synthesis of lipids in deeper soil (Matsumoto et al., 2007; Gleixner, 2013) rather than the stabilization of
microbial necromass (Kramer and Gleixner, 2008). Here we examine the 14C characteristics of Glycerol Dialkyl Glycerol Tetraethers (GDGTs) – characteristic membrane lipids
of microorganisms that are ubiquitous in terrestrial and aqueous environments (Schouten et al., 2013). GDGTs are subdivided signatures of fatty acids and phospholipid-fatty acids (PLFAs), established indicators for plant and microbial-derived C, suggest
30
active microbial re-synthesis of lipids in deeper soil (Matsumoto et al., 2007; Gleixner, 2013) rather than the stabilization of
microbial necromass (Kramer and Gleixner, 2008). Here we examine the 14C characteristics of Glycerol Dialkyl Glycerol Tetraethers (GDGTs) – characteristic membrane lipids active microbial re-synthesis of lipids in deeper soil (Matsumoto et al., 2007; Gleixner, 2013) rather than the stabilization of
microbial necromass (Kramer and Gleixner, 2008). 1
Introduction Despite their rapid adoption by biogeochemists and paleoclimatologists as valuable molecular tracers and proxies of carbon
45
source and environmental conditions, there are numerous aspects regarding their production, turnover and fate that remain
enigmatic. In particular, despite their ubiquity in soils and other environmental matrices, the biological precursors, metabolic
processes and physiological drivers giving rise to brGDGT signatures observed in terrestrial and aquatic systems remain poorly
constrained. In this context, natural abundance-level radiocarbon measurements of these compounds may provide a valuable Despite their rapid adoption by biogeochemists and paleoclimatologists as valuable molecular tracers and proxies of carbon
45
source and environmental conditions, there are numerous aspects regarding their production, turnover and fate that remain
enigmatic. In particular, despite their ubiquity in soils and other environmental matrices, the biological precursors, metabolic
processes and physiological drivers giving rise to brGDGT signatures observed in terrestrial and aquatic systems remain poorly
constrained. In this context, natural abundance-level radiocarbon measurements of these compounds may provide a valuable approach to better understand their source(s) and turnover rates, while also shedding light on processes that influence their
50
abundance and distribution. Prior 14C-based studies of GDGTs have primarily focused on the isoprenoid compounds in marine waters and sediments
(Pearson et al., 2001; Smittenberg et al., 2004; Ingalls et al., 2006; Mollenhauer et al., 2007, 2008; Shah et al., 2008). The only
reported investigation of branched GDGT 14C characteristics in lake sediments (Birkholz et al., 2013) yielded ∆14C values
than expected based on the depositional age of the sediment that were lower than those of the depositional age of the sediment
55 approach to better understand their source(s) and turnover rates, while also shedding light on processes that influence their
50
abundance and distribution. Prior 14C-based studies of GDGTs have primarily focused on the isoprenoid compounds in marine waters and sediments
(Pearson et al., 2001; Smittenberg et al., 2004; Ingalls et al., 2006; Mollenhauer et al., 2007, 2008; Shah et al., 2008). The only
reported investigation of branched GDGT 14C characteristics in lake sediments (Birkholz et al., 2013) yielded ∆14C values than expected based on the depositional age of the sediment. that were lower than those of the depositional age of the sediment,
55 2 2 https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. although the causes of this pre-aged signal was not established. 2.1
Study Site At each site soil cores were sampled following protocols implemented as part of the LWF sampling program (van der Voort
et al., 2016). These soil composites have been previously analyzed for radiocarbon signatures of organic carbon in bulk soil and
soil density fractions, as well as in specific alkanes and fatty acids (Van der Voort et al., 2017), which allows the comparison
of the isoprenoid and branched GDGTs with other biomarkers and operationally-defined soil carbon pools. 80 of the isoprenoid and branched GDGTs with other biomarkers and operationally-defined soil carbon pools. 80 of the isoprenoid and branched GDGTs with other biomarkers and operationally-defined soil carbon pools. 80 1
Introduction Although soils are considered a major source of brGDGTs to
aquatic systems, the 14C signatures of brGDGTs in soils has not been determined. Thus, we presently lack crucial information
concerning both the production and cycling of this distinctive group of microbial lipids in the context of soil C cycling, as well
as the implications for their uses as molecular tracers and proxies. although the causes of this pre-aged signal was not established. Although soils are considered a major source of brGDGTs to
aquatic systems, the 14C signatures of brGDGTs in soils has not been determined. Thus, we presently lack crucial information
concerning both the production and cycling of this distinctive group of microbial lipids in the context of soil C cycling, as well
as the implications for their uses as molecular tracers and proxies. In the present study, we used molecular-level natural-abundance 14C measurements to constrain the provenance and turnover
60
of GDGTs in soils. We developed and rigorously tested a preparative high performance liquid chromatography (HPLC) method
to isolate both isoprenoid and branched GDGTs from soils (and sediments) for subsequent small-scale radiocarbon analysis
by accelerator mass spectrometry (AMS). We then applied this method to samples from two well-studied sub-alpine Swiss
soil profiles in order to shed light on their origin and stability. As unequivocal markers of microbial contributions to soils, the
GDGTs provide an opportunity to assess the stability and turnover of microbial biomass in soils. 65 60 2.1
Study Site Soils were sampled by taking soil cores at two forested sites in Switzerland, one near Lausanne (46.5838◦N, 6.6580◦E, 800 m
asl), and the other one close to Beatenberg (46.7003◦N, 7.7623◦E, 1490 m asl). Both sites are both part of the Long-term Forest
Ecosystem Research (LWF) network (Innes, 1995) maintained by the Swiss Federal Institute for Forest, Snow and Landscape
70
Research (WSL). Soils were sampled by taking soil cores at two forested sites in Switzerland, one near Lausanne (46.5838◦N, 6.6580◦E, 800 m
asl), and the other one close to Beatenberg (46.7003◦N, 7.7623◦E, 1490 m asl). Both sites are both part of the Long-term Forest
Ecosystem Research (LWF) network (Innes, 1995) maintained by the Swiss Federal Institute for Forest, Snow and Landscape
70
Research (WSL). The subalpine soil from the site at Beatenberg is a Podzol which has a thick organic layer followed by a 10 cm A-horizon, and
carbonate-free sandstone as parent material. The MAT at the site is 4.6◦C and the pH rises from 3.7 to 4.4 with increasing soil
depth The second soil from the Swiss Plateau close to Lausanne is a Cambisol developed on top of a carbonate containing Research (WSL). The subalpine soil from the site at Beatenberg is a Podzol which has a thick organic layer followed by a 10 cm A-horizon, and
carbonate-free sandstone as parent material. The MAT at the site is 4.6◦C and the pH rises from 3.7 to 4.4 with increasing soil
depth. The second soil from the Swiss Plateau close to Lausanne is a Cambisol developed on top of a carbonate-containing moraine. Its A-Horizon extends to 50 cm with a total soil depth of 3 m. Here, the MAT is 7.6◦C, the pH is slightly higher
75
compared to the Beatenberg soil and largely invariant (4.6 to 4.5) to a depth of 80 cm (Walthert, 2003). At each site soil cores were sampled following protocols implemented as part of the LWF sampling program (van der Voort
et al., 2016). These soil composites have been previously analyzed for radiocarbon signatures of organic carbon in bulk soil and moraine. Its A-Horizon extends to 50 cm with a total soil depth of 3 m. Here, the MAT is 7.6◦C, the pH is slightly higher
75
compared to the Beatenberg soil and largely invariant (4.6 to 4.5) to a depth of 80 cm (Walthert, 2003). 2.2
Reference Materials for Method Validation Evaluation of the isolation method involved assessment of the purity of separated fractions (i.e., potential interference from
other than the desired compounds) and determination of the amount and isotopic composition external contamination intro-
duced in course of the preparation sequence. For the latter, composites of different topsoil samples (0-5 cm) from a 30 x 30
m grassland area in central Switzerland (5.9% C, bulk soil organic carbon (OC) F 14C = 1.155) and a Rhineland lignite from
85 85 3 3 https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. the early Miocene (Heumann and Litt, 2002)(62.3% OC, bulk F 14C = 0.003) were used for assessment and validation with
regard to contamination. the early Miocene (Heumann and Litt, 2002)(62.3% OC, bulk F 14C = 0.003) were used for assessment and validation with
regard to contamination. 2.3
GDGT isolation for radiocarbon measurement Despite the relative ease of detection of GDGTs using modern HPLC-mass spectrometry (MS) techniques, one challenge
in the radiocarbon analysis of GDGTs in soil and sediment samples is their low abundance, with ambient concentrations of
90
brGDGTs and isoGDGTs that are typically in the range of 10 to 1000 ng gdw−1 and 1 to 100 ng gdw−1 (grams dry weight)
soil, respectively (Weijers et al., 2006b). A separation of individual GDGTs of the soil samples used in this study would
require on average 3000 g of soil to reach the minimum recommended mass (∼15 µg C) for high-precision compound-specific
radiocarbon analysis (Haghipour et al., 2018). As extracting several kg of material is impractical, focused instead on pooled
isolation and 14C measurement of isoprenoid GDGTs and branched GDGTs, respectively, at the compound class level, due
95
to the common putative biological percursors and biosynthetic formation pathways for each compound class (Schouten et al.,
2013). For this study, the pooling of the GDGTs reduced the required initial sample size to a maximum of 500 gdw of soil. The
extraction and purification of the compounds prior to HPLC analysis purification of the compounds prior to HPLC analysis
followed a procedure that is similar to that applied to samples processed for quantification of GDGTs (Freymond et al., 2017). In brief:
100 Despite the relative ease of detection of GDGTs using modern HPLC-mass spectrometry (MS) techniques, one challenge
in the radiocarbon analysis of GDGTs in soil and sediment samples is their low abundance, with ambient concentrations of
90
brGDGTs and isoGDGTs that are typically in the range of 10 to 1000 ng gdw−1 and 1 to 100 ng gdw−1 (grams dry weight)
soil, respectively (Weijers et al., 2006b). A separation of individual GDGTs of the soil samples used in this study would
require on average 3000 g of soil to reach the minimum recommended mass (∼15 µg C) for high-precision compound-specific
radiocarbon analysis (Haghipour et al., 2018). As extracting several kg of material is impractical, focused instead on pooled isolation and 14C measurement of isoprenoid GDGTs and branched GDGTs, respectively, at the compound class level, due
95
to the common putative biological percursors and biosynthetic formation pathways for each compound class (Schouten et al.,
2013). For this study, the pooling of the GDGTs reduced the required initial sample size to a maximum of 500 gdw of soil. 2.3
GDGT isolation for radiocarbon measurement The
extraction and purification of the compounds prior to HPLC analysis purification of the compounds prior to HPLC analysis
followed a procedure that is similar to that applied to samples processed for quantification of GDGTs (Freymond et al., 2017). In brief:
100
lipids were extracted from dried soil samples using a microwave (CEM MARS 5) or an Energized Dispersive Guided Extraction
(CEM EDGE) system. No difference in performance was observed for the different extraction systems. Samples were processed
in batches of roughly 15 to 20 g of material. For microwave extraction the samples were transferred to the extraction vessels
and covered by a dichloromethane (DCM):methanol (MeOH) 9:1 (v/v, 25 ml) solvent mixture. Extraction temperature was In brief:
100
lipids were extracted from dried soil samples using a microwave (CEM MARS 5) or an Energized Dispersive Guided Extraction
(CEM EDGE) system. No difference in performance was observed for the different extraction systems. Samples were processed
in batches of roughly 15 to 20 g of material. For microwave extraction the samples were transferred to the extraction vessels
and covered by a dichloromethane (DCM):methanol (MeOH) 9:1 (v/v, 25 ml) solvent mixture. Extraction temperature was programmed to ramp to 100◦C in 35 min and is subsequently held for 20 min. For EDGE extraction 25 ml DCM:MeOH 9:1
105
(v/v) was used for extraction at 110◦C for 2 min and subsequent rinsing with 15 ml followed by a second extraction with 5 ml
of solvent at 100◦C and rinsing with 35 ml. The process was repeated on additional sample batches to yield sufficient quantities
of the target lipid compounds for 14C analysis. Pooled extracts were then dried under nitrogen flow. After the addition of 5
ml MilliQ water with NaCl the neutral phase was back-extracted with hexane (Hex) and separated on a 1% deactivated silica programmed to ramp to 100◦C in 35 min and is subsequently held for 20 min. For EDGE extraction 25 ml DCM:MeOH 9:1
105
(v/v) was used for extraction at 110◦C for 2 min and subsequent rinsing with 15 ml followed by a second extraction with 5 ml
of solvent at 100◦C and rinsing with 35 ml. The process was repeated on additional sample batches to yield sufficient quantities
of the target lipid compounds for 14C analysis. Pooled extracts were then dried under nitrogen flow. 2.3
GDGT isolation for radiocarbon measurement After the addition of 5
ml MilliQ water with NaCl the neutral phase was back-extracted with hexane (Hex) and separated on a 1% deactivated silica column into apolar and polar fractions with Hex:DCM 9:1 (v/v) and DCM:MeOH 1:1 (v/v) respectively. Polar fractions were
110
dried under N2, then re-dissolved in Hex:2-propanol (IPA) 99:1 (v/v) and passed over 0.45 µm PTFE filters. A portion of
the polar fraction was set aside (1%) and an internal C46 GDGT standard (Huguet et al., 2006) was added to this aliquot to
determine GDGT concentrations. Polar fractions were separated on an Agilent 1260 HPLC system coupled to an Agilent 1260 fraction collector Separation was Polar fractions were separated on an Agilent 1260 HPLC-system coupled to an Agilent 1260 fraction collector. Separation was
achieved on two Waters Aquity UHPLC HEB Hilic columns (1.7 µm, 2.1 x 150 mm) connected in series and preceded by a
115
2.1 x 5 mm guard column (Hopmans et al., 2016). The columns were heated to 45◦C and the flow rate set to 0.2 ml min−1. For
the first 25 min, compounds elute isocratically with a solvent mixture of 18% Hex:IPA 9:1 (v/v) (solvent A) and 82% hexane
(solvent B). For the next 15 min the proportion of solvent B was decreased linearly to 65% followed by a linear gradient
to 0% solvent B in 20 min. The total runtime of one injection hence sums up to 60 min followed by 20 min reequilibration achieved on two Waters Aquity UHPLC HEB Hilic columns (1.7 µm, 2.1 x 150 mm) connected in series and preceded by a
115
2.1 x 5 mm guard column (Hopmans et al., 2016). The columns were heated to 45◦C and the flow rate set to 0.2 ml min−1. For
the first 25 min, compounds elute isocratically with a solvent mixture of 18% Hex:IPA 9:1 (v/v) (solvent A) and 82% hexane
(solvent B). For the next 15 min the proportion of solvent B was decreased linearly to 65% followed by a linear gradient
to 0% solvent B in 20 min. The total runtime of one injection hence sums up to 60 min followed by 20 min reequilibration 4 4 https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. with 82% solvent B. 2.3
GDGT isolation for radiocarbon measurement The fraction collection is solely based on retention times with the isoprenoid fraction being collected
120
from 14.5 to 26 min and the branched fraction from 33 to 43 min (Figure 1). The retention time is recurrently monitored to
avoid undetected drifts. The injection volume is set to 15 µl corresponding to total GDGT amounts of 100 to 300 ng ml−1. Each sample was injected 10 times and fractions were pooled afterwards. The isolated compound classes and the subset of the
initial polar fraction set aside previously were analyzed for purity and quantification using the same HPLC system coupled to a quadrupole mass spectrometer (Agilent 6130) according to Hopmans et al. (2016). The isolated fractions were dried and
125
transferred into 0.025 ml tin capsules (Elementar 03951620). The capsules containing each sample were measured using an
elemental analyzer coupled to a gas-ion-source equipped accelerator mass spectrometer (EA-AMS) (Haghipour et al., 2018) at
the laboratory of Ion Beam Physics at ETH Zürich (Synal et al., 2007; Ruff et al., 2007). In all cases, sample sizes were > 15
µg C. to a quadrupole mass spectrometer (Agilent 6130) according to Hopmans et al. (2016). The isolated fractions were dried and
125
transferred into 0.025 ml tin capsules (Elementar 03951620). The capsules containing each sample were measured using an
elemental analyzer coupled to a gas-ion-source equipped accelerator mass spectrometer (EA-AMS) (Haghipour et al., 2018) at
the laboratory of Ion Beam Physics at ETH Zürich (Synal et al., 2007; Ruff et al., 2007). In all cases, sample sizes were > 15
µg C. to a quadrupole mass spectrometer (Agilent 6130) according to Hopmans et al. (2016). The isolated fractions were dried and
125
transferred into 0.025 ml tin capsules (Elementar 03951620). The capsules containing each sample were measured using an
elemental analyzer coupled to a gas-ion-source equipped accelerator mass spectrometer (EA-AMS) (Haghipour et al., 2018) at
the laboratory of Ion Beam Physics at ETH Zürich (Synal et al., 2007; Ruff et al., 2007). In all cases, sample sizes were > 15
µg C. 2.4
Soil turnover model
130 The ∆14C of atmospheric decomposition rate constants of each pool k1 and k2, as well as the relative size of the two pools. The ∆14C of atmospheric
CO2 was taken from Hua et al. (2013) from 1950 to 1986 and from Hammer and Levin (2017) for the years thereafter. 140 CO2 was taken from Hua et al. (2013) from 1950 to 1986 and from Hammer and Levin (2017) for the years thereafter. 140 CO2 was taken from Hua et al. (2013) from 1950 to 1986 and from Hammer and Levin
140 2.4
Soil turnover model
130 Turnover times of the individual compounds are calculated based on a steady state two-pool box model (e.g., Trumbore et al.,
1996; Torn et al., 2009; Schrumpf and Kaiser, 2015; van der Voort et al., 2019). This model assumes two homogenous pools
with a first-order decay rate, a fast-cycling and a passive pool. For each of the pools the F 14C is calculated independently
(equation 1), where F 14Cpool(t) is the radiocarbon signal of the respective pool in the sampling year t, lag is the number of Turnover times of the individual compounds are calculated based on a steady state two-pool box m Turnover times of the individual compounds are calculated based on a steady state two-pool box model (e.g., Trumbore et al.,
1996; Torn et al., 2009; Schrumpf and Kaiser, 2015; van der Voort et al., 2019). This model assumes two homogenous pools
with a first-order decay rate, a fast-cycling and a passive pool. For each of the pools the F 14C is calculated independently
(equation 1), where F 14Cpool(t) is the radiocarbon signal of the respective pool in the sampling year t, lag is the number of years between CO2 fixation in plants and plant litter entering the soil, λ is the radioactive decay of 14C (1/8267 years), and
135
kpool is the decomposition rate constant. years between CO2 fixation in plants and plant litter entering the soil, λ is the radioactive decay of 14C (1/8267 years), and
135
kpool is the decomposition rate constant. F 14Cpool(t) = F 14Catm(t−lag) ∗kpool + F 14Cpool(t−1) ∗(1 −kpool −λ) F 14Cpool(t) = F 14Catm(t−lag) ∗kpool + F 14Cpool(t−1) ∗(1 −kpool −λ)
(1) ag) ∗kpool + F 14Cpool(t−1) ∗(1 −kpool −λ)
(1) (1) The fraction-weighted sum of the F 14C of each of the pools is the modelled F 14C of the sample and depends on the
decomposition rate constants of each pool k1 and k2, as well as the relative size of the two pools. The ∆14C of atmospheric
CO2 was taken from Hua et al. (2013) from 1950 to 1986 and from Hammer and Levin (2017) for the years thereafter. 140 The fraction-weighted sum of the F 14C of each of the pools is the modelled F 14C of the sample and depends on the
decomposition rate constants of each pool k1 and k2, as well as the relative size of the two pools. 3.1
Method Validation The recommended sample size to reach a precision <5% varies depending on the age of the sample. For samples with a
radiocarbon age < 1800 years (F 14C > 0.8) a size of 20 µg C is sufficient to reach the desired precision, while samples older
165
than 6000 years (F 14C < 0.5) require at least 50 µg C. These uncertainties are taken into account when considering the GDGT
14C results for the soil samples measured in this study. 3.1
Method Validation The best fit for F 14Cc and mc to match the observed F 14Cm for both sets of measurements is calculated according to Hag
155 to match the observed F 14Cm for both sets of measurements is calculated according to Haghipour et al. (2018). 155 The blank assessment (Figure 2) yields a contamination of 2.62 ± 0.79µg C with a fraction modern of 0.59 ± 0.18, which
is in range of previously determined contamination introduced by HPLC separation of lipids (e.g., Shah and Pearson, 2007;
Birkholz et al., 2013). The impact of the constant contamination decreases as the sample mass increases. Therefore, the limit The blank assessment (Figure 2) yields a contamination of 2.62 ± 0.79µg C with a fraction modern of 0.59 ± 0.18, which
is in range of previously determined contamination introduced by HPLC separation of lipids (e.g., Shah and Pearson, 2007;
Birkholz et al., 2013). The impact of the constant contamination decreases as the sample mass increases. Therefore, the limit towards large carbon masses of the fitted curve is equivalent to the radiocarbon signal of the sample unaffected by extraneously
160
introduced carbon. For both samples, this limit and hence the compound F 14C differs from the bulk F 14C of the initial
material. In the topsoil reference the compounds are depleted in radiocarbon (F 14C = 0.94) with respect to the source, in the
lignite the GDGTs are enriched (F 14C = 0.06). towards large carbon masses of the fitted curve is equivalent to the radiocarbon signal of the sample unaffected by extraneously
160
introduced carbon. For both samples, this limit and hence the compound F 14C differs from the bulk F 14C of the initial
material. In the topsoil reference the compounds are depleted in radiocarbon (F 14C = 0.94) with respect to the source, in the
lignite the GDGTs are enriched (F 14C = 0.06). The recommended sample size to reach a precision <5% varies depending on the age of the sample. For samples with a
radiocarbon age < 1800 years (F 14C > 0.8) a size of 20 µg C is sufficient to reach the desired precision, while samples older
165
than 6000 years (F 14C < 0.5) require at least 50 µg C. These uncertainties are taken into account when considering the GDGT
14C results for the soil samples measured in this study. 3.1
Method Validation Repetitive preparation of samples with 10 injections each reveals a recovery efficiency of 0.85 ± 0.05. Analysis of isolated
fractions on a quadrupole mass spectrometer operated in scan mode (Agilent 6130) for all masses between m/z 500 and 1500
reveals that more than 95% of compounds in either fraction are comprised of masses assigned to GDGTs (Figure 1). 145 5 https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. The extraneous contamination added in the preparatory process is assumed to be of constant mass mc and radiocarbon
signature F 14Cc. Therefore, the measured signal F 14Cm is a mixture of the sample and the contaminant according to equation
2: The extraneous contamination added in the preparatory process is assumed to be of constant mass mc and radiocarbon
signature F 14Cc. Therefore, the measured signal F 14Cm is a mixture of the sample and the contaminant according to equation F 14Cm = F 14Cs ∗ms + F 14Cc ∗mc
ms + mc
(2) F 14Cm = F 14Cs ∗ms + F 14Cc ∗mc
ms + mc (2) (2) F 14Cs and ms are the true radiocarbon signal and carbon mass of the sample. The measured F 14Cm changes depending
150
on the mass of the sample, as smaller masses are more strongly affected by the constant contamination. We assume that in
samples with a bulk radiocarbon signal that is either completely modern or does not contain any 14C at all the compound-
specific radiocarbon value is similar to the bulk. Therefore, a radiocarbon-modern sample, i.e., the topsoil composite, and the
radiocarbon-dead lignite were prepared and measured repeatedly with different concentrations. The best fit for F 14Cc and mc F 14Cs and ms are the true radiocarbon signal and carbon mass of the sample. The measured F 14Cm changes depending
150
on the mass of the sample, as smaller masses are more strongly affected by the constant contamination. We assume that in
samples with a bulk radiocarbon signal that is either completely modern or does not contain any 14C at all the compound-
specific radiocarbon value is similar to the bulk. Therefore, a radiocarbon-modern sample, i.e., the topsoil composite, and the
radiocarbon-dead lignite were prepared and measured repeatedly with different concentrations. 3.3
Radiocarbon variations The GDGT
∆14C values are also more depleted than those of DOC and short-chain (C16−22) FA in the soil, but are bracketed by n-C27 n-
alkane and n-C29 fatty acid ∆14C values in the deepest soil section (Van der Voort et al., 2017). The ∆14C values of the density -20 and -7‰ at 0 to 5 cm to -441 and -310 ‰ at 60 to 80 cm, respectively. While ∆14C values of bulk OC and GDGTs parallel
195
one another closely in the Beatenberg soil, GDGT ∆14C values are systematically lower than bulk OC and at each depth
interval in the Lausanne profile, with the difference between GDGTs and bulk OC ranging from -105 to -200 ‰. The GDGT
∆14C values are also more depleted than those of DOC and short-chain (C16−22) FA in the soil, but are bracketed by n-C27 n-
alkane and n-C29 fatty acid ∆14C values in the deepest soil section (Van der Voort et al., 2017). The ∆14C values of the density fractions from the same soil samples were also measured by Van der Voort et al. (2017). The low density fraction corresponds
200
to the free particulate organic carbon (free POC) and the high density fraction is interpreted as mineral-associated POC. Both
fractions do not differ by more than 40 ‰ in the top 20 cm of either soil profile, but in the lowest depth interval the fractions
diverge, with markedly lower ∆14C values the for high density fraction, and values similar to DOC for the free POC fraction. In both soils, the iso and brGDGTs exhibit similar or lower ∆14C values than the high density fraction, mineral-associated
organic matter fraction. 205 fractions from the same soil samples were also measured by Van der Voort et al. (2017). The low density fraction corresponds
200
to the free particulate organic carbon (free POC) and the high density fraction is interpreted as mineral-associated POC. Both
fractions do not differ by more than 40 ‰ in the top 20 cm of either soil profile, but in the lowest depth interval the fractions
diverge, with markedly lower ∆14C values the for high density fraction, and values similar to DOC for the free POC fraction. In both soils, the iso and brGDGTs exhibit similar or lower ∆14C values than the high density fraction, mineral-associated
organic matter fraction. 205 80 cm. The relative abundance of the individual brGDGTs also changes with soil depth, as reflected in the MBT’5Me and CBT’ ratio
(De Jonge et al., 2014). The MBT’5Me index does not exhibit significant variability in either soil profile, while the CBT’ index
increases with soil depth, especially in the Lausanne soil indicating a shift towards 6-methylated GDGTs (Figure 3). 180 3.2
Vertical Distributions of GDGTs In the pre-alpine soil from Beatenberg, concentrations of GDGTs are generally highest in In the pre-alpine soil from Beatenberg, concentrations of GDGTs are generally highest in the topsoil, where the isoprenoid
and branched GDGTs are 10 and 38 µg gdw−1 respectively, whereas corresponding concentrations in the top soil layer of
170
the Lausanne soil are much lower, 0.6 and 2 µg gdw−1, respectively (Figure 3). The concentration of both groups of GDGTs
decreases sharply with increasing soil depths, with approximately ten times the abundance of isoGDGTs and brGDGTs in the
top 5 cm than a few centimeters below. In contrast, isoprenoid and branched GDGTs concentrations normalized to organic
carbon (OC) content increase with depth in the Beatenberg soil from 47µg gOC−1 for isoGDGTs and from 175 µg gOC−1 for and branched GDGTs are 10 and 38 µg gdw−1 respectively, whereas corresponding concentrations in the top soil layer of
170
the Lausanne soil are much lower, 0.6 and 2 µg gdw−1, respectively (Figure 3). The concentration of both groups of GDGTs
decreases sharply with increasing soil depths, with approximately ten times the abundance of isoGDGTs and brGDGTs in the
top 5 cm than a few centimeters below. In contrast, isoprenoid and branched GDGTs concentrations normalized to organic
carbon (OC) content increase with depth in the Beatenberg soil from 47µg gOC−1 for isoGDGTs and from 175 µg gOC−1 for brGDGTs in the top 5 cm to 273 µg gOC−1 and 80 µg gOC−1, respectively, between 20 and 40 cm depth. In the Lausanne soil
175
profile, the OC-normalized isoprenoid and branched concentrations drop from 10 µg gOC−1 and 39 µg gOC−1, respectively,
in the top 5 cm to 4 and 13 µg gOC−1 between 10 and 20 cm depth, and then increase to 14 and 12 µg gOC−1 between 60 and 6 80 cm. The relative abundance of the individual brGDGTs also changes with soil depth, as reflected in the MBT’5Me and CBT’ ratio
(De Jonge et al., 2014). The MBT’5Me index does not exhibit significant variability in either soil profile, while the CBT’ index
180
increases with soil depth, especially in the Lausanne soil indicating a shift towards 6-methylated GDGTs (Figure 3). 3 3
R di
b
i ti
https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. 3.3
Radiocarbon variations The GDGT fractions prepared for AMS measurement contained between 30 and 80 µg C, except for the brGDGTs in the 10
to 20 cm depth interval in Beatenberg and the iso- and brGDGTs from 60 to 80 cm the Lausanne soil, which range between
15 and 20 µg C. The results of the radiocarbon measurements are shown in Figure 4, together with previously reported 14C
185
data for other soil carbon constituents (Van der Voort et al., 2017) . In the Beatenberg profile, radiocarbon signatures of both
isoprenoid and branched GDGTs closely follow the bulk OC with ∆14C values of -23 and -30 ‰, respectively, in the top 5 cm,
and decreasing to -241 and -196 ‰ in the 20 to 40 cm depth interval, respectively. This contrasts with dissolved organic carbon
(DOC), which exhibits 14C-enriched and relatively invariant ∆14C values throughout the soil profile (73 ‰ in the topsoil to
24 ‰ in the deeper soil)(Figure 4). Overall, ∆14C values of both groups of GDGTs are similar to those of a C29 n-alkane and
190
the long-chained (>C26) n-alkanoic fatty acids (FA), but differ sharply from those of shorter-chained (C16-C22 measured on The GDGT fractions prepared for AMS measurement contained between 30 and 80 µg C, except for the brGDGTs in the 10
to 20 cm depth interval in Beatenberg and the iso- and brGDGTs from 60 to 80 cm the Lausanne soil, which range between 15 and 20 µg C. The results of the radiocarbon measurements are shown in Figure 4, together with previously reported 14C
185
data for other soil carbon constituents (Van der Voort et al., 2017) . In the Beatenberg profile, radiocarbon signatures of both
isoprenoid and branched GDGTs closely follow the bulk OC with ∆14C values of -23 and -30 ‰, respectively, in the top 5 cm,
and decreasing to -241 and -196 ‰ in the 20 to 40 cm depth interval, respectively. This contrasts with dissolved organic carbon
(DOC), which exhibits 14C-enriched and relatively invariant ∆14C values throughout the soil profile (73 ‰ in the topsoil to 15 and 20 µg C. The results of the radiocarbon measurements are shown in Figure 4, together with previously reported 14C
185
data for other soil carbon constituents (Van der Voort et al., 2017) . 3.3
Radiocarbon variations In the Beatenberg profile, radiocarbon signatures of both
isoprenoid and branched GDGTs closely follow the bulk OC with ∆14C values of -23 and -30 ‰, respectively, in the top 5 cm,
and decreasing to -241 and -196 ‰ in the 20 to 40 cm depth interval, respectively. This contrasts with dissolved organic carbon
(DOC), which exhibits 14C-enriched and relatively invariant ∆14C values throughout the soil profile (73 ‰ in the topsoil to 24 ‰ in the deeper soil)(Figure 4). Overall, ∆14C values of both groups of GDGTs are similar to those of a C29 n-alkane and
190
the long-chained (>C26) n-alkanoic fatty acids (FA), but differ sharply from those of shorter-chained (C16-C22 measured on
the same samples (Van der Voort et al., 2017). The latter never reach ∆14C values lower than -90 ‰ in the soils, resulting in
an offset between short-chained fatty acids and the other analyzed compounds that show stronger decreases with soil depth. Similar patterns exist in the Lausanne profile: The ∆14C values in isoprenoid and branched GDGTs decrease with depth from 24 ‰ in the deeper soil)(Figure 4). Overall, ∆14C values of both groups of GDGTs are similar to those of a C29 n-alkane and
190
the long-chained (>C26) n-alkanoic fatty acids (FA), but differ sharply from those of shorter-chained (C16-C22 measured on
the same samples (Van der Voort et al., 2017). The latter never reach ∆14C values lower than -90 ‰ in the soils, resulting in
an offset between short-chained fatty acids and the other analyzed compounds that show stronger decreases with soil depth. Similar patterns exist in the Lausanne profile: The ∆14C values in isoprenoid and branched GDGTs decrease with depth from Similar patterns exist in the Lausanne profile: The ∆
C values in isoprenoid and branched GDGTs decrease with depth from
-20 and -7‰ at 0 to 5 cm to -441 and -310 ‰ at 60 to 80 cm, respectively. While ∆14C values of bulk OC and GDGTs parallel
195
one another closely in the Beatenberg soil, GDGT ∆14C values are systematically lower than bulk OC and at each depth
interval in the Lausanne profile, with the difference between GDGTs and bulk OC ranging from -105 to -200 ‰. 3.4
Radiocarbon derived turnover times of GDGTs Turnover times of the compounds are calculated based on a two-pool model that requires three parameters to be fitted: the
turnover time of the fast-cycling pool, the turnover time of the passive pool and the proportion of the fast-cycling pool. As only
one radiocarbon measurement per compound and depth interval is available, two of the parameters need to be estimated, while
one can be fitted accordingly. We use the proportion of the labile low-density fraction of the samples (Van der Voort et al.,
210 7 7 https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. 2017) to constrain the size of the fast-cycling pool. The turnover time of the fast-cycling pool can be estimated accordingly as
the single-pool turnover time of the light fraction. Alternatively, the GDGT turnover in topsoil based on stable carbon isotopes
has been shown to be similar to short-chain fatty acids (Weijers et al., 2010; Huguet et al., 2017). Thus, the turnover time of
these compounds based on a single-pool box model can also be used to constrain turnover time of the fast-cycling pool of 2017) to constrain the size of the fast-cycling pool. The turnover time of the fast-cycling pool can be estimated accordingly as
the single-pool turnover time of the light fraction. Alternatively, the GDGT turnover in topsoil based on stable carbon isotopes
has been shown to be similar to short-chain fatty acids (Weijers et al., 2010; Huguet et al., 2017). Thus, the turnover time of
these compounds based on a single-pool box model can also be used to constrain turnover time of the fast-cycling pool of GDGTs. For simplicity, a lag-term addressing the time between atmospheric carbon fixation and input into the soil is not used,
215
as it is shorter than a decade (Solly et al., 2018) and its potential influence is hence already covered by the range of the turnover
time estimates of the fast pool. The low ∆14C values of GDGTs in the deeper soil intervals result in a turnover time of the
passive GDGT pool in the order of 1400 to 2000 years between 10 and 20 cm depth and 2000 up to 6000 years in the lowest
depth interval in either soil (Table 1). These results are insensitive to changes of either the turnover time and the size of the 215 GDGTs. 3.4
Radiocarbon derived turnover times of GDGTs For simplicity, a lag-term addressing the time between atmospheric carbon fixation and input into the soil is not used,
215
as it is shorter than a decade (Solly et al., 2018) and its potential influence is hence already covered by the range of the turnover
time estimates of the fast pool. The low ∆14C values of GDGTs in the deeper soil intervals result in a turnover time of the
passive GDGT pool in the order of 1400 to 2000 years between 10 and 20 cm depth and 2000 up to 6000 years in the lowest
depth interval in either soil (Table 1). These results are insensitive to changes of either the turnover time and the size of the labile pool: A change of ±10 % of the proportion of the fast pool or ±500 years of the turnover time of the fast pool results in
220
a maximum change of 10 % of the overall GDGT turnover time in the two lower depth intervals in either soil. Thus, despite the
uncertainties in the estimation of the proportion of the pools and the turnover time of the fast pool, the turnover time of both
groups of GDGTs clearly exceeds a millenium. labile pool: A change of ±10 % of the proportion of the fast pool or ±500 years of the turnover time of the fast pool results in
220
a maximum change of 10 % of the overall GDGT turnover time in the two lower depth intervals in either soil. Thus, despite the
uncertainties in the estimation of the proportion of the pools and the turnover time of the fast pool, the turnover time of both
groups of GDGTs clearly exceeds a millenium. labile pool: A change of ±10 % of the proportion of the fast pool or ±500 years of the turnover time of the fast pool results in
220
a maximum change of 10 % of the overall GDGT turnover time in the two lower depth intervals in either soil. Thus, despite the
uncertainties in the estimation of the proportion of the pools and the turnover time of the fast pool, the turnover time of both
groups of GDGTs clearly exceeds a millenium. 4.1
Efficacy of GDGT isolation and 14C measurement protocol
225 Compared to prior methods to achieve individual isoGDGT separation by HPLC (Smittenberg et al., 2002; Ingalls et al., 2006),
the introduced method isolates GDGTs only at the compound-class level, hence potential radiocarbon variations among GDGT
isomers are not discernable. However, previous analyses of stable carbon isotopic as well as radiocarbon analysis of GDGTs
on a molecular level do not show significant differences between the individual isoprenoid or branched GDGTs, respectively (e.g., Ingalls et al., 2006; Shah et al., 2008; Oppermann et al., 2010; Weber et al., 2015). This implies similar metabolisms
230
for brGDGT-producing organisms and also for microbial communities that synthesize isoGDGTs. Consequently, pooling of
isomers within a compound class according to their respective microbial domain (bacteria, archaea) seems reasonable, par-
ticularly given the practical constraints imposed by their low abundance in many terrestrial (and aquatic) environments. The
introduced method requires only a single normal-phase isolation step using the same columns that are used for quantification of (e.g., Ingalls et al., 2006; Shah et al., 2008; Oppermann et al., 2010; Weber et al., 2015). This implies similar metabolisms
230
for brGDGT-producing organisms and also for microbial communities that synthesize isoGDGTs. Consequently, pooling of
isomers within a compound class according to their respective microbial domain (bacteria, archaea) seems reasonable, par-
ticularly given the practical constraints imposed by their low abundance in many terrestrial (and aquatic) environments. The
introduced method requires only a single normal-phase isolation step using the same columns that are used for quantification of GDGTs (Hopmans et al., 2016), minimizing the time required for sample preparation and without extensive adjustments to the
235
analytical HPLC set-up. The calculated contamination is in range of the blank assessment by Ingalls et al. (2006), but higher
than the extraneous carbon observed by Birkholz et al. (2013). However, the blank assessment in Birkholz et al. (2013) is
based only on a modern non-GDGT standard (cholesterol), potentially leading to an underestimation of the sample preparation
blank. GDGTs (Hopmans et al., 2016), minimizing the time required for sample preparation and without extensive adjustments to the
235
analytical HPLC set-up. The calculated contamination is in range of the blank assessment by Ingalls et al. (2006), but higher
than the extraneous carbon observed by Birkholz et al. (2013). However, the blank assessment in Birkholz et al. (2013) is
based only on a modern non-GDGT standard (cholesterol), potentially leading to an underestimation of the sample preparation
blank. 4.1
Efficacy of GDGT isolation and 14C measurement protocol
225 The GDGT-specific ∆14C values of the top soil and lignite samples used as "modern" and "fossil" endmembers for blank
240
assessment did not yield values that fully matched those expected given their age. In case of the soil, different ∆14C values of
the GDGTs compared to bulk OC are to be expected due to the heterogeneous nature of soil organic matter, however for lignite
sample that is of geologic age (> 30 Ma), all components would be expected to be radiocarbon-dead. A preliminary batch of The GDGT-specific ∆14C values of the top soil and lignite samples used as "modern" and "fossil" endmembers for blank
240
assessment did not yield values that fully matched those expected given their age. In case of the soil, different ∆14C values of
the GDGTs compared to bulk OC are to be expected due to the heterogeneous nature of soil organic matter, however for lignite
sample that is of geologic age (> 30 Ma), all components would be expected to be radiocarbon-dead. A preliminary batch of 8 https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. lignite that was extracted yielded 18 µg C of isoGDGTs and 48 µg C of brGDGTs, with corresponding ∆14C values of the
resulting isolated compounds of of -960‰ and -980‰, respectively. The second batch of lignite used to assess constant con-
245
tamination was prepared 4 months later and shows ∆14C values consistently higher than -950‰ (figure 2). This shift towards
higher ∆14C values likely reflects contamination resulting from sample-to-sample carry-over on the HPLC. Although this is
adressed in the blank assessment, this highlights the importance of repeated blank assessment in order to control for variations
in carry-over and other potential sources of contamination (e.g., column bleed) over time. Careful assessment of compound
purity is also important to ensure robust isotopic determination. 250 4.2
Radiocarbon constraints on the origin and turnover of GDGTs in soils Consequently, the 14C contents of iso- and brGDGTs
should reflect that of the carbon source of their biological precursors. IsoGDGTs are known to be produced by Thaumarcheota
and Euryarcheota (Schouten et al., 2013). The specific microbes that produce brGDGTs are yet to be identified, there is
strong evidence that the precursor organisms are heterotrophic bacteria (Pancost and Damsté, 2003; Weijers et al., 2010),
265
with Acidobacteria amongst the candidate phyla (Damsté et al., 2018). For heterotrophic bacteria, potential carbon sources
include DOC leached from the organic layer, exudates from root systems or organic matter that has accumulated during soil
development. The activity of soil microbial communities has often been assayed using phospholipid-fatty acids (PLFAs), as
phospholipids are only found in living cells and thus serve as biomarkers for viable microbial communities (e.g., Tunlid and
White, 1991). Compound-specific radiocarbon analyses of PLFAs have shown that soil microbes can use a variety of carbon
270
sources, including “older” SOM (Kramer and Gleixner, 2006). However, root-derived C seems a dominant food source of
heterotrophic microbial communities in temperate deciduous forest soils (Kramer et al., 2010) and this has been inferred to
be a likely substrate for the producers of brGDGTs (Huguet et al., 2013). The turnover time of root carbon is on the order
of decades at most (Gill and Jackson, 2000; Gaudinski et al., 2001; Solly et al., 2018)), and thus the old ages and long
turnover times of GDGTs observed in both soils analyzed in this study cannot be explained by the uptake of root-derived C. 275
Accessible, labile carbon pools in the investigated soil profiles are represented by DOC and the light density fraction. These
appear to be preferably used by microbial communities as evidenced by the 14C-enriched values of short-chain (<C24) fatty strong evidence that the precursor organisms are heterotrophic bacteria (Pancost and Damsté, 2003; Weijers et al., 2010),
265
with Acidobacteria amongst the candidate phyla (Damsté et al., 2018). For heterotrophic bacteria, potential carbon sources
include DOC leached from the organic layer, exudates from root systems or organic matter that has accumulated during soil
development. 4.2
Radiocarbon constraints on the origin and turnover of GDGTs in soils Our study reveals low ∆14C values, with corresponding radiocarbon ages of up to 6000 years for GDGTs in forested soils. These 14C characteristics are similar to those of the mineral-associated OM (from density fractionation), as well as long-chain,
higher plant wax-derived n-fatty acids and n-alkanes. As GDGTs are microbial membrane lipids, these findings reveal the
presence of 14C-depleted, millennial age microbial residues as a component of organic matter in deeper soils. There are two
255
possible pathways leading to these old apparent radiocarbon ages: (1) active GDGT-producing heterotrophic soil microbial
communities in deeper soils are utilizing pre-aged SOM as a carbon source, and accrue this signal with continuous community Our study reveals low ∆14C values, with corresponding radiocarbon ages of up to 6000 years for GDGTs in forested soils. These 14C characteristics are similar to those of the mineral-associated OM (from density fractionation), as well as long-chain,
higher plant wax-derived n-fatty acids and n-alkanes. As GDGTs are microbial membrane lipids, these findings reveal the g
p
y
p
,
g
presence of 14C-depleted, millennial age microbial residues as a component of organic matter in deeper soils. There are two
255
possible pathways leading to these old apparent radiocarbon ages: (1) active GDGT-producing heterotrophic soil microbial
communities in deeper soils are utilizing pre-aged SOM as a carbon source, and accrue this signal with continuous community
turnover. Alternatively, (2) upon cell death these microbial lipids are stabilized for millenia, likely via interaction with soil
minerals. We first consider the first explanation: The ∆14C values of living organisms, and their constituent lipids, directly reflect that of their metabolic carbon source
260
as, unlike stable isotopes, they are impervious to biological fractionation effects (Ingalls and Pearson, 2005). Upon death
of the organism, radioactive decay leads to depletion in 14C contents. Consequently, the 14C contents of iso- and brGDGTs
should reflect that of the carbon source of their biological precursors. IsoGDGTs are known to be produced by Thaumarcheota
and Euryarcheota (Schouten et al., 2013). The specific microbes that produce brGDGTs are yet to be identified, there is The ∆14C values of living organisms, and their constituent lipids, directly reflect that of their metabolic carbon source
260
as, unlike stable isotopes, they are impervious to biological fractionation effects (Ingalls and Pearson, 2005). Upon death
of the organism, radioactive decay leads to depletion in 14C contents. 4.2
Radiocarbon constraints on the origin and turnover of GDGTs in soils The activity of soil microbial communities has often been assayed using phospholipid-fatty acids (PLFAs), as
phospholipids are only found in living cells and thus serve as biomarkers for viable microbial communities (e.g., Tunlid and strong evidence that the precursor organisms are heterotrophic bacteria (Pancost and Damsté, 2003; Weijers et al., 2010),
265
with Acidobacteria amongst the candidate phyla (Damsté et al., 2018). For heterotrophic bacteria, potential carbon sources
include DOC leached from the organic layer, exudates from root systems or organic matter that has accumulated during soil
development. The activity of soil microbial communities has often been assayed using phospholipid-fatty acids (PLFAs), as
phospholipids are only found in living cells and thus serve as biomarkers for viable microbial communities (e.g., Tunlid and White, 1991). Compound-specific radiocarbon analyses of PLFAs have shown that soil microbes can use a variety of carbon
270
sources, including “older” SOM (Kramer and Gleixner, 2006). However, root-derived C seems a dominant food source of
heterotrophic microbial communities in temperate deciduous forest soils (Kramer et al., 2010) and this has been inferred to
be a likely substrate for the producers of brGDGTs (Huguet et al., 2013). The turnover time of root carbon is on the order
of decades at most (Gill and Jackson, 2000; Gaudinski et al., 2001; Solly et al., 2018)), and thus the old ages and long turnover times of GDGTs observed in both soils analyzed in this study cannot be explained by the uptake of root-derived C. 275
Accessible, labile carbon pools in the investigated soil profiles are represented by DOC and the light density fraction. These
appear to be preferably used by microbial communities as evidenced by the 14C-enriched values of short-chain (<C24) fatty 9 https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. acids that likely reflect active microbial communities (Figure 4, 5). The markedly lower ∆14C values of both isoGDGTs
and brGDGTs at depth would require that both groups of precursor organisms, i.e., Archaea and Bacteria, occupy specific
niches using metabolic strategies that enable them to utilize stabilized, aged carbon. The precursor organisms of isoGDGTs
280
in soils are known to be mainly comprised of crenarchaeota, i.e., chemoautotrophic nitrifiers using soil CO2 as substrate
(Leininger et al., 2006; Urich et al., 2008; Weijers et al., 2010; Damsté et al., 2012) and acetotrophic methanogens (Weijers
et al., 2010). 4.2
Radiocarbon constraints on the origin and turnover of GDGTs in soils Contributions from the latter organisms in the studies soils are likely minor as the soils are not strictly anaerobic
(Walthert, 2003)). This is also supported by GDGT-0/Crenarchaeol ratios, that differ sharply from those in soils and sediments dominated by methanogens (Blaga et al., 2009; Weijers et al., 2010; Naeher et al., 2014). Soil-respired CO2 has relatively
285
high ∆14C values (Gaudinski et al., 2000; Liu et al., 2006), and thus it seems highly unlikely that 14C-depleted signatures
of isoGDGTs in the deeper soils results from metabolism of an old C substrate by active soil microbial communities. By
analogy, the 14C-depleted characteristics of brGDGTs is difficult to reconcile with heterotrophic consumption of pre-aged C. Overall, the contrasting metabolisms of the GDGT precursor organisms (primarily autotrophy for isoGDGTs and heterotrophy dominated by methanogens (Blaga et al., 2009; Weijers et al., 2010; Naeher et al., 2014). Soil-respired CO2 has relatively
285
high ∆14C values (Gaudinski et al., 2000; Liu et al., 2006), and thus it seems highly unlikely that 14C-depleted signatures
of isoGDGTs in the deeper soils results from metabolism of an old C substrate by active soil microbial communities. By
analogy, the 14C-depleted characteristics of brGDGTs is difficult to reconcile with heterotrophic consumption of pre-aged C. Overall, the contrasting metabolisms of the GDGT precursor organisms (primarily autotrophy for isoGDGTs and heterotrophy for brGDGTs), yet similar (and low) ∆14C values for both compound classes, argue against an origin of the GDGT signals
290
from microbial growth at depth.We therefore conclude that uptake of pre-aged carbon by active soil microbial communities is
unlikely to be the cause for the 14C-depleted GDGT signatures. We next consider the long-term stabilization microbially-derived carbon as the source of 14C-depleted GDGT signatures. This implies that microbial residues persist in soils for millennia, lending support to emerging concepts that microbial necro- for brGDGTs), yet similar (and low) ∆14C values for both compound classes, argue against an origin of the GDGT signals
290
from microbial growth at depth.We therefore conclude that uptake of pre-aged carbon by active soil microbial communities is
unlikely to be the cause for the 14C-depleted GDGT signatures. We next consider the long-term stabilization microbially-derived carbon as the source of 14C-depleted GDGT signatures. This implies that microbial residues persist in soils for millennia, lending support to emerging concepts that microbial necro- mass comprises an important component of older SOM (e.g., Lehmann and Kleber, 2015; Liang et al., 2017). 4.2
Radiocarbon constraints on the origin and turnover of GDGTs in soils Long-term
295
persistence of GDGTs could arise from their stabilization by soil minerals at greater soil depths. The amphiphilic nature of
lipids such as GDGTs, with both polar and hydrophobic components, promotes the association with mineral surfaces, and
therefore may afford physical protection from degradation (Jandl et al., 2004; Kleber et al., 2007; von Lützow et al., 2008;
Van der Voort et al., 2017). By comparison, in surface soils with high organic matter contents and less availability of reactive mineral surfaces, GDGTs are continuously produced and degraded, which results in a younger mean radiocarbon age and evi-
300
dence for turnover on decadal timescales (Weijers et al., 2010). This explanation agrees with conceptual models of soil organic
matter dynamics whereby older SOM in deeper soils primarily consists of microbial metabolites that are stabilized by their
interaction with mineral surfaces (Schmidt et al., 2011; Lehmann and Kleber, 2015). Given the structural resemblance between
brGDGTs and isoGDGTs, and hence similar propensity to associate with mineral surfaces , we consider this a more likely mineral surfaces, GDGTs are continuously produced and degraded, which results in a younger mean radiocarbon age and evi-
300
dence for turnover on decadal timescales (Weijers et al., 2010). This explanation agrees with conceptual models of soil organic
matter dynamics whereby older SOM in deeper soils primarily consists of microbial metabolites that are stabilized by their
interaction with mineral surfaces (Schmidt et al., 2011; Lehmann and Kleber, 2015). Given the structural resemblance between
brGDGTs and isoGDGTs, and hence similar propensity to associate with mineral surfaces , we consider this a more likely explanation for their similarly old 14C ages than “niche metabolisms” of different precursor organisms. The older GDGT age
305
in the the Cambisol at Lausanne compared to the subalpine Podzol at Beatenberg with a bleached eluvial horizon also supports
this conclusion. The higher contents of clay and highly reactive amorphous Fe and Al-oxides and hydroxides of the former
(Table 2) are known to play a key role in the sorptive stabilization of SOM (Kaiser and Guggenberger, 2003; Kleber et al.,
2007) explanation for their similarly old 14C ages than “niche metabolisms” of different precursor organisms. The older GDGT age
305
in the the Cambisol at Lausanne compared to the subalpine Podzol at Beatenberg with a bleached eluvial horizon also supports
this conclusion. 4.3
Implications for application of GDGTs as molecular proxies and soil tracer biomolecul In addition to the insights into soil carbon turnover, the observed 14C signatures of GDGTs in the two soil profiles carry impli-
315
cations for their application as proxies of environmental conditions and as tracers of soil carbon input to aquatic environments. Several studies have shown that soils comprise a significant, and often the dominant, component of terrestrial organic carbon
exported in the suspended load of rivers to lake and ocean sediments (e.g., Tao et al., 2015; Vonk et al., 2019; Hein et al., 2020). Prior analyses of branched GDGTs in sedimentary archives have revealed older GDGT ages than depositional ages (Smitten- berg et al., 2005; Birkholz et al., 2013) that may reflect intermittent storage during transport or export of deeper mineral soil
320
carbon suggesting a lag between production and deposition. Our findings suggest that this may be a consequence of protracted
storage in and mobilization from deeper mineral soils. This reinforces the value of brGDGTs as tracers of soil carbon, but
implies that much of the signal may be sourced from deeper soil layers, with corresponding GDGT proxy signals reflecting
environmental conditions at the time that they were microbially produced. 5
Conclusions
325 We modified and validated a normal-phase HPLC method to isolate isoprenoid and branched GDGTs at the compound class
level for radiocarbon analysis. Although further refinements in the method would be desirable, this new approach yields reliable
GDGT 14C measurements on sample sizes > 20 µg C that have enabled novel questions to be addressed concerning the
provenance and turnover of this key suite of microbial lipids. In addition to its application to questions of soil C cycling, the
li
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f hi streamlined method opens up new opportunities to further explore the biogeochemical and paleoclimate significance of this
330
intriguing yet enigmatic class of lipids. Application of the method to depth profiles for two well-studied sub-alpine soil profiles in Switzerland reveals a marked
decrease in 14C contents of both isoGDGTs and brGDGTs with depth, with resulting model estimates for GDGT turnover
times of 2000 to 6000 years in deeper mineral soils. These old ages for archaeal and bacterial membrane lipids provides compelling evidence for stabilization of microbial necromass in soils that contributes to the long-term C storage. Through
335
comparison with parallel 14C data for soil density fractions and other hydrophobic lipid biomarkers, we attribute the stability
of GDGTs to protection via association with reactive mineral surfaces underlining the crucial role of microbial processes in
soil C cycling and stabilization. Our findings also provide motivation for further work to validate our interpretations and assess the broader significance of the Our findings also provide motivation for further work to validate our interpretations and assess the broader significance of the
current limited suite of observations. For example, comparison of the proportions and isotopic signatures of intact polar lipid
340
GDGTs relative to the “core” lipids measured here could shed light on the significance active GDGT-producing communities
residing at a specific soil depth versus remnants of past microbial activity (necromass). Furthermore, while concentrations of
isoprenoid as well as branched GDGTs commonly decrease with increasing soil depth (Huguet et al., 2010; Yamamoto et al., current limited suite of observations. For example, comparison of the proportions and isotopic signatures of intact polar lipid
340
GDGTs relative to the “core” lipids measured here could shed light on the significance active GDGT-producing communities
residing at a specific soil depth versus remnants of past microbial activity (necromass). 4.2
Radiocarbon constraints on the origin and turnover of GDGTs in soils The higher contents of clay and highly reactive amorphous Fe and Al-oxides and hydroxides of the former
(Table 2) are known to play a key role in the sorptive stabilization of SOM (Kaiser and Guggenberger, 2003; Kleber et al.,
2007) Overall, 14C characteristics of iso and brGDGTs and the inferred turnover times that are far longer than those of discrete POM
310
(free light density fraction) and signature lipids of active microbial communities (short-chained fatty acids), but similar to those Overall, 14C characteristics of iso and brGDGTs and the inferred turnover times that are far longer than those of discrete POM
310
(free light density fraction) and signature lipids of active microbial communities (short-chained fatty acids), but similar to those Overall, 14C characteristics of iso and brGDGTs and the inferred turnover times that are far longer than those of discrete POM
310
(free light density fraction) and signature lipids of active microbial communities (short-chained fatty acids), but similar to those 10 https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. of plant-derived long-chain n-alkanes and fatty acids (Figure 5), serve as strong evidence for the presence of mineral-stabilized
microbial necromass in the studied forested mineral soils. of plant-derived long-chain n-alkanes and fatty acids (Figure 5), serve as strong evidence for the presence of mineral-stabilized
microbial necromass in the studied forested mineral soils. 5
Conclusions
325 Furthermore, while concentrations of
isoprenoid as well as branched GDGTs commonly decrease with increasing soil depth (Huguet et al., 2010; Yamamoto et al., 11 https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. 2016; Gocke et al., 2017), subsurface maxima in iso- and brGDGT concentrations have also been reported (Huguet et al.,
2010; Yamamoto et al., 2016)), potentially indicating depth-localized GDGT production. Future 14C analysis of GDGTs in
345
soil profiles that exhibit such sub-surface concentrations peak would be informative and provide context for our observations
in the two Swiss soil profiles. Further insights into the provenance and turnover of brGDGTs might be gained from in-depth
assessment of molecular distributions and associated proxy indices (De Jonge et al., 2014), that may reflect changes in current
or past microbial communities, or imply differences in susceptibility to degradation. Despite the presence of GDGTs as trace
constituents of SOM their unequivocal microbial origin distinctive chemical structures and environmental properties that
350 constituents of SOM, their unequivocal microbial origin, distinctive chemical structures, and environmental properties that
350
their distributions encode render them powerful tracer compounds and molecular proxies. Here, we demonstrate that when
also constrained with natural abundance 14C , these compounds provide a new window into the role of microorganisms in soil
carbon cycling. constituents of SOM, their unequivocal microbial origin, distinctive chemical structures, and environmental properties that
350
their distributions encode render them powerful tracer compounds and molecular proxies. Here, we demonstrate that when
also constrained with natural abundance 14C , these compounds provide a new window into the role of microorganisms in soil
carbon cycling. Code and data availability. The data set and script for the turnover model used in this study is available at https://doi.org/10.3929/ethz-b-
000430425
55 355 000430425 Author contributions. HG, ML and TE conceptualized the study, HG and DM designed the method, HG isolated the compounds and wrote
the turnover model, NH performed radiocarbon measurements, TSvdV provided data, HG, FH, ML and TE interpreted the data, HG prepared
the manuscript with contributions from all co-authors. References Ahrens, B., Braakhekke, M. C., Guggenberger, G., Schrumpf, M., and Reichstein, M.: Contribution of sorption, DOC transport and microbial
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360
thank Urs Overhoff and Markus Neuroth from the RWE Power AG for the provisioning of the lignite sample, and Marco Griepentrog
and Cindy de Jonge for helpful discussions. We thank the Laboratory for Ion Beam Physics (ETH) for support with the Accelerator Mass
Spectrometry measurements. Acknowledgements. This study was funded by the Swiss National Science Foundation (“CAPS-LOCK2”&“CAPS-LOCK3”; #184865). We
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thank Urs Overhoff and Markus Neuroth from the RWE Power AG for the provisioning of the lignite sample, and Marco Griepentrog
and Cindy de Jonge for helpful discussions. We thank the Laboratory for Ion Beam Physics (ETH) for support with the Accelerator Mass
Spectrometry measurements. Acknowledgements. This study was funded by the Swiss National Science Foundation (“CAPS-LOCK2”&“CAPS-LOCK3”; #184865). We
360
thank Urs Overhoff and Markus Neuroth from the RWE Power AG for the provisioning of the lignite sample, and Marco Griepentrog
and Cindy de Jonge for helpful discussions. We thank the Laboratory for Ion Beam Physics (ETH) for support with the Accelerator Mass
Spectrometry measurements. 12 https://doi.org/10.5194/bg-2020-308
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b
C
S h
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1999, Tech. rep., ETH Zurich, 2003. Weber, Y., De Jonge, C., Rijpstra, W. I. C., Hopmans, E. C., Stadnitskaia, A., Schubert, C. J., Lehmann, M. F., Weber, Y., De Jonge, C., Rijpstra, W. I. C., Hopmans, E. C., Stadnitskaia, A., Schubert, C. J., Lehmann, M. F., Damste, J. S. S., and Niemann,
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paleothermometer in paleosols, Geomicrobiology Journal, 33, 98–109, 2016. e b a e
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Postdepositional effect on the estimation of air temperature, Quaternary international, 397, 380–391, 2016. Yamamoto, Y., Ajioka, T., and Yamamoto, M.: Climate reconstruction based on GDGT based proxies in a paleosol sequence in Japan:
Postdepositional effect on the estimation of air temperature, Quaternary international, 397, 380–391, 2016. Yang, H., Pancost, R. D., Jia, C., and Xie, S.: The response of archaeal tetraether membrane lipids in surface soils to temperature: a potential
paleothermometer in paleosols, Geomicrobiology Journal, 33, 98–109, 2016. Yang, H., Pancost, R. D., Jia, C., and Xie, S.: The response of archaeal tetraether membrane lipids in surface soils to temperature: a potential
paleothermometer in paleosols, Geomicrobiology Journal, 33, 98–109, 2016. Yang, H., Pancost, R. D., Jia, C., and Xie, S.: The response of archaeal tetraether membrane lipids in surface soils to temperature: a potential
paleothermometer in paleosols, Geomicrobiology Journal, 33, 98–109, 2016. Zimmermann, S., Luster, J., Blaser, P., Walthert, L., and Lüscher, P.: Waldböden der Schweiz, Band 3, Regionen Mittelland und Voralpen,
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Swiss Federal Research Institute WSL, Hep Verlag Bern, Switzerland, 2006. 18 https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. 1260 1280 1300 1320
m/z
10
30
50
percentage
1000
1040
1080
m/z
10
20
30
percentage
10
15
20
25
30
35
40
45
50
isoprenoid GDGTs
branched GDGTs
(a)
(b)
(c)
t [min]
Cren
GDGT-0
GDGT-I
II
III
Figure 1. HPLC mass chromatograms (m/z 500 - 1500) of GDGTs from a composition topsoil sample. a) sample before GDGT isolation,
b) the separated isoprenoid fraction and its composite mass spectrum (GDGT-3, GDGT-2 and GDGT-1 from left to right between Crenarcheol
(Cren) and GDGT-0 are not labelled), c) the separated branched fraction and its composite mass spectrum (GDGT-I, II and III, corresponding
to tetra-, penta- and hexamethylated GDGTs are labelled)(For molecular structures see Figure A1) 1260 1280 1300 1320
m/z
10
30
50
percentage
isoprenoid GDGTs
branched GDGTs
(a)
(b)
Cren
GDGT-0 (a) (b) 1000
1040
1080
m/z
10
20
30
percentage
10
15
20
25
30
35
40
45
50
(c)
t [min]
GDGT-I
II
III (c) 10 Figure 1. References HPLC mass chromatograms (m/z 500 - 1500) of GDGTs from a composition topsoil sample. a) sample before GDGT isolation,
b) the separated isoprenoid fraction and its composite mass spectrum (GDGT-3, GDGT-2 and GDGT-1 from left to right between Crenarcheol
(Cren) and GDGT-0 are not labelled), c) the separated branched fraction and its composite mass spectrum (GDGT-I, II and III, corresponding
to tetra-, penta- and hexamethylated GDGTs are labelled)(For molecular structures see Figure A1) 19 https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. 0
10
20
30
mass C [µg]
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
F14C
0
10
20
30
40
50
mass C [µg]
0.7
0.8
0.9
1
1.1
F14C
(a)
(b)
Figure 2. Blank assessment associated with GDGT isolation. a) The F 14C dead standard (lignite) tracing a modern constant contamination
of 1.55 µg C, b) The F 14C modern standard (topsoil composite) tracing a radiocarbon dead constant contamination of 1.07 µg C. The solid
line indicates the best fit of contamination mass and radiocarbon signal for both sets of samples considered jointly, the grey dotted lines show
the 1σ error range 0
10
20
30
mass C [µg]
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
F14C
(a) 0
10
20
30
40
50
mass C [µg]
0.7
0.8
0.9
1
1.1
F14C
(b) (b) Figure 2. Blank assessment associated with GDGT isolation. a) The F 14C dead standard (lignite) tracing a modern constant contamination
of 1.55 µg C, b) The F 14C modern standard (topsoil composite) tracing a radiocarbon dead constant contamination of 1.07 µg C. The solid
line indicates the best fit of contamination mass and radiocarbon signal for both sets of samples considered jointly, the grey dotted lines show
the 1σ error range 20 https://doi.org/10.5194/bg-2020-308
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Beatenberg
−300
−200
−100
0
100
Δ14C [‰]
depth [cm]
−400
−200
0
200
Δ14C [‰]
depth [cm]
60
to
80
10
to
20
to
5
to
5
10
to
20
20
to
40
(a)
isoGDGTs
brGDGTs
bulk SOM
free POM
MAOM
DOC
FAC16 −C22
FAC26
FAC28
Alkane
(b)
Figure 4. Radiocarbon content, expressed as ∆14C of organic matter fractions and compounds in Lausanne and Beatenberg soil
profiles. In both soils (Lausanne - A, Beatenberg - B) the radiocarbon contents of both branched and isoprenoid GDGTs decrease to a similar
degree to that of bulk SOM in each profile. The measured n-alkane in the Beatenberg soil is C29, in the Lausanne soil the n-C27 homologue
was analyzed. Alkane, n-fatty acid (FA), free particulate organic matter (POM), mineral-associated organic matter (MAOM) and dissolved
organic carbon (DOC) ∆14C values are taken from Van der Voort et al. (2017). DOC was not measured in the same intervals as the other
parameters, but at 0, 15, 50 and 80 cm, and at 0 and 30 cm depth in the Lausanne and Beatenberg soils, respectively. If error bars are not
visible, then the uncertainty is smaller than the symbol size. Beatenberg −400
−200
0
200
Δ14C [‰]
depth [cm]
60
to
80
10
to
20
to
5
(a)
isoGDGTs
brGDGTs
bulk SOM
free POM
MAOM
DOC
FAC16 −C22
FAC26
FAC28
Alkane −300
−200
−100
0
100
Δ14C [‰]
depth [cm]
to
5
10
to
20
20
to
40
(b) (b) nt, expressed as ∆14C of organic matter fractions and compounds in Lausanne and Beatenberg soil Figure 4. Radiocarbon content, expressed as ∆14C of organic matter fractions and compounds in L gu e
. ad oca bo
co te t, e p essed as
C o o ga
c
atte
act o s a d co
pou ds
ausa
e a d
eate be g so
profiles. In both soils (Lausanne - A, Beatenberg - B) the radiocarbon contents of both branched and isoprenoid GDGTs decrease to a similar
degree to that of bulk SOM in each profile. The measured n-alkane in the Beatenberg soil is C29, in the Lausanne soil the n-C27 homologue
was analyzed. https://doi.org/10.5194/bg-2020-308
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c⃝Author(s) 2020. CC BY 4.0 License. Lausanne
Beatenberg
0
10
20
30
40
concentration [µg/g soil]
depth [cm]
isoGDGTs
brGDGTs
0.0
0.5
1.0
1.5
2.0
concentration [µg/g soil]
depth [cm]
60
to
80
10
to
20
to
5
to
5
10
to
20
20
to
40
0.56
0.60
0.64
−1.46 −1.42 −1.38
CBT'
0.49
0.51
0.53
MBT’5ME
−1.6 −1.4 −1.2 −1.0
CBT'
MBT’5ME
60-80
10-20
0-5
20-40
10-20
0-5
(a)
(b)
Figure 3. BrGDGT abundances and parameter ratios in Lausanne and Beatenberg soil profiles. In both soils (a - Lausanne, b - Beat-
enberg) the concentration of the samples per dry weight decreases rapidly with depth. The inner plots show the brGDGT-derived MBT’5ME
(orange) and the CBT’ (blue) indices in the respective soil. While the MBT’5ME does not vary a lot with depth, the CBT’ increases signifi-
cantly with depth in the Lausanne soil. 0.0
0.5
1.0
1.5
2.0
concentration [µg/g soil]
depth [cm]
60
to
80
10
to
20
to
5
0.49
0.51
0.53
MBT’5ME
−1.6 −1.4 −1.2 −1.0
CBT'
60-80
10-20
0-5
(a) 0
10
20
30
40
concentration [µg/g soil]
depth [cm]
s
to
5
10
to
20
20
to
40
0.56
0.60
0.64
−1.46 −1.42 −1.38
CBT'
MBT’5ME
20-40
10-20
0-5
(b) (b) Figure 3. BrGDGT abundances and parameter ratios in Lausanne and Beatenberg soil profiles. In both soils (a - Lausanne, b - Beat-
enberg) the concentration of the samples per dry weight decreases rapidly with depth. The inner plots show the brGDGT-derived MBT’5ME
(orange) and the CBT’ (blue) indices in the respective soil. While the MBT’5ME does not vary a lot with depth, the CBT’ increases signifi-
cantly with depth in the Lausanne soil. 21 https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. Alkane, n-fatty acid (FA), free particulate organic matter (POM), mineral-associated organic matter (MAOM) and dissolved
organic carbon (DOC) ∆14C values are taken from Van der Voort et al. (2017). DOC was not measured in the same intervals as the other
parameters, but at 0, 15, 50 and 80 cm, and at 0 and 30 cm depth in the Lausanne and Beatenberg soils, respectively. If error bars are not
visible, then the uncertainty is smaller than the symbol size. 22 https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. −200
−150
−100
−50
0
50
100
bulk Δ14C [‰]
−500
−400
−300
−200
−100
0
100
200
compound Δ14C [‰]
isoGDGTs
brGDGTs
FAC16 −C22
FAC26
FAC28
Alkane
free POM
MAOM
longer turnover time than bulk SOM
shorter turnover time than bulk SOM
Beatenberg
Lausanne
Figure 5. Relationship between ∆14C values of specific components and bulk SOM in Beatenberg and Lausanne soil profiles. The
different analyzed compounds and density fractions (Van der Voort et al., 2017) cover a greater range of ∆14C values with soil depth as
reflected in the ageing of the bulk OC in the respective soil. The dotted line represents equal compound and bulk ∆14C. Two groups are
discernable: those with ∆14C values higher than the bulk OC, and thus with a more rapid turnover (in most samples, this includes short-
chain (C16-C22) FA and the low density fraction (free POM) and those with lower ∆14C values implying longer turnover times (including
long-chain n-alkanes (in the Beatenberg soil C29-alkane, in the Lausanne soil the C27), and fatty acids (C26, C28 FA) and the GDGTs. −200
−150
−100
−50
0
50
100
bulk Δ14C [‰]
−500
−400
−300
−200
−100
0
100
200
compound Δ14C [‰]
isoGDGTs
brGDGTs
FAC16 −C22
FAC26
FAC28
Alkane
free POM
MAOM
longer turnover time than bulk SOM
shorter turnover time than bulk SOM
Beatenberg
Lausanne Figure 5. Relationship between ∆14C values of specific components and bulk SOM in Beatenberg and Lausanne soil profiles. The
different analyzed compounds and density fractions (Van der Voort et al., 2017) cover a greater range of ∆14C values with soil depth as
reflected in the ageing of the bulk OC in the respective soil. The dotted line represents equal compound and bulk ∆14C. Two groups are
discernable: those with ∆14C values higher than the bulk OC, and thus with a more rapid turnover (in most samples, this includes short-
chain (C16-C22) FA and the low density fraction (free POM) and those with lower ∆14C values implying longer turnover times (including
long-chain n-alkanes (in the Beatenberg soil C29-alkane, in the Lausanne soil the C27), and fatty acids (C26, C28 FA) and the GDGTs. 23 https://doi.org/10.5194/bg-2020-308
Preprint. https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. Table 1. Turnover times of isoprenoid and branched GDGTs.Turnover times are based on the single-pool turnover time of the light
fraction (fPOM) or the short-chain fatty acids (SCFA - in brackets) as approximation of the fast pool. Table 1. Turnover times of isoprenoid and branched GDGTs.Turnover times are based on the single-pool turnover time of the
fraction (fPOM) or the short-chain fatty acids (SCFA - in brackets) as approximation of the fast pool. fast pool
turnover times fPOM (SCFA) [years]
loc
depth
proportion
fast pool
isoGDGT
brGDGT
Bb
0-5cm
0.897
350
(340)
710
(740)
440
(450)
10-20cm
0.193
890
(920)
1400
(1400)
1600
(1600)
20-40cm
0.115
350
(760)
2800
(2700)
2100
(2000)
Ln
0-5cm
0.162
46
(33)
350
(360)
540
(550)
10-20cm
0.113
240
(240)
2000
(2000)
1400
(1400)
60-80cm
0.087
1200
(300)
3600
(3700)
5900
(6100) Table 1. Turnover times of isoprenoid and branched GDGTs.Turnover times are based on the single-pool turnover time of the light
fraction (fPOM) or the short-chain fatty acids (SCFA - in brackets) as approximation of the fast pool. 24 24 https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. Table 2. Soil properties related to the stability of soil organic matter. CEC (effective cation exchange capacity), Fed (dithionite-extractable
iron), Feo and Alo (oxalate-extractable iron and aluminum),Fep and Alp (pyrophosphate-extractable iron and aluminum) as well as sand, silt
and clay content are provided by Zimmermann et al. (2006)
CEC
Fed
Feo
Fep
Alo
Alp
sand
silt
clay
loc
depth
[mmolC kg−1]
[ppm]
[ppm]
[ppm]
[ppm]
[ppm]
[%]
[%]
[%]
Bb
0-5cm
50
1047
684
505
538
571
84
7
8
10-20cm
15
na
133
99
280
268
83
15
3
20-40cm
37
5770
2183
2062
880
1713
80
14
6
Ln
0-5cm
77
6187
3356
2900
1861
1304
62
25
13
10-20cm
61
6493
3039
2310
2030
1430
51
31
18
60-80cm
59
5840
2095
732
1156
791
57
27
16 Table 2. Soil properties related to the stability of soil organic matter. CEC (effective cation exchange capacity), Fed (dithionite-extractable
iron), Feo and Alo (oxalate-extractable iron and aluminum),Fep and Alp (pyrophosphate-extractable iron and aluminum) as well as sand, silt
and clay content are provided by Zimmermann et al. (2006) Table 2. Soil properties related to the stability of soil organic matter. https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. 1292
1292’
1302
1300
1298
1296
Crenarchaeol
Crenarchaeol’
GDGT - 0
GDGT - 1
GDGT - 2
GDGT - 3
Ia
Ib
Ic
IIa / IIa’
IIb / IIb’
IIc / IIc’
IIIa / IIIa’
IIIb / IIIb’
IIIc / IIIc’
1022
1020
1018
1036
1034
1032
1050
1048
1046
isoprenoid GDGTs
branched GDGTs
Name m/z
Name 6-methyl isomer m/z
Supplementary Figure 1: Structures of GDGTs analyzed in this study. Figure A1. Molecular structures of GDGTs analyzed in this study branched GDGTs isoprenoid GDGTs isoprenoid GDGTs Ia
Ib
Ic
IIa / IIa’
IIb / IIb’
IIc / IIc’
IIIa / IIIa’
IIIb / IIIb’
IIIc / IIIc’
1022
1020
1018
1036
1034
1032
1050
1048
1046
branched GDGTs
Name 6-methyl isomer m/z 1292
1292’
1302
1300
1298
1296
Crenarchaeol
Crenarchaeol’
GDGT - 0
GDGT - 1
GDGT - 2
GDGT - 3
Name m/z 6-methyl isomer m/z 6-methyl isomer m/z Name https://doi.org/10.5194/bg-2020-308
Preprint. Discussion started: 26 August 2020
c⃝Author(s) 2020. CC BY 4.0 License. CEC (effective cation exchange capacity), Fed (dithionite-extractable
iron), Feo and Alo (oxalate-extractable iron and aluminum),Fep and Alp (pyrophosphate-extractable iron and aluminum) as well as sand, silt
and clay content are provided by Zimmermann et al. (2006) 25 Name Supplementary Figure 1: Structures of GDGTs analyzed in this study. Figure A1. Molecular structures of GDGTs analyzed in this study 26
|
https://openalex.org/W2623029357
|
https://uu.diva-portal.org/smash/get/diva2:1038767/FULLTEXT01
|
English
| null |
Search for Higgs and<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mi>Z</mml:mi></mml:math>Boson Decays to<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mrow><mml:mi>ϕ</mml:mi><mml:mi>γ</mml:mi></mml:mrow></mml:math>with the ATLAS Detector
|
Physical review letters
| 2,016
|
cc-by
| 20,667
|
DOI: 10.1103/PhysRevLett.117.111802 Rare decays of the 125 GeV Higgs boson [1,2] H to a
light meson and a photon γ have been suggested to present
one viable probe of the Yukawa coupling of the Higgs
boson to light (u, d, s) quarks [3–5]. While the Standard
Model (SM) predicts these couplings to be small, sub-
stantial modifications are predicted in several scenarios
beyond the SM, which include the minimal flavor violation
framework [6], the Froggatt-Nielsen mechanism [7], the
Higgs-dependent
Yukawa
couplings
model
[8],
the
Randall-Sundrum family of models [9], and the possibility
of the Higgs boson being a composite pseudo-Goldstone
boson [10]. The light-quark Yukawa couplings are almost
entirely unconstrained by existing data and the large
multijet background at the Large Hadron Collider (LHC)
severely inhibits the study of such couplings with inclusive
H →q¯q decays. The decay of the Higgs boson to a ϕ
meson and a photon would give access to the strange-quark
Yukawa coupling and to potential deviations from the SM
prediction. The
expected
SM
branching
fraction
is
BðH →ϕγÞ ¼ ð2.3 0.1Þ × 10−6 [4], and no direct exper-
imental information about this decay mode currently exists. The analogous rare decays of the Higgs boson to a heavy
quarkonium state and a photon offer sensitivity to the
charm- and bottom-quark Yukawa couplings [11–13]. The
Higgs boson decays to J=ψγ and ϒγ have already been
searched for by the ATLAS Collaboration [14]. The former
decay mode has also been searched for by the CMS
Collaboration [15]. fractions much smaller than for Higgs boson decays to the
same final state. The most precise prediction for the SM
branching fraction is BðZ →ϕγÞ ¼ ð1.17 0.08Þ × 10−8
[16]. The decay Z →ϕγ has not yet been observed and is
not well constrained by existing measurements of Z boson
decays. This Letter describes a search for Higgs and Z boson
decays to the exclusive final state ϕγ. The decay
ϕ →KþK−is used to reconstruct the ϕ meson. The search
is performed with a sample of pp collision data corre-
sponding to an integrated luminosity of 2.7 fb−1 recorded
at a center-of-mass energy
ffiffiffis
p ¼ 13 TeV with the ATLAS
detector, described in detail in Ref. [18]. Published by the American Physical Society under the terms of
the Creative Commons Attribution 3.0 License. Further distri-
bution of this work must maintain attribution to the author(s) and
the published article’s title, journal citation, and DOI. week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) *Full author list given at the end of the article. DOI: 10.1103/PhysRevLett.117.111802 Higgs boson production is modeled using the POWHEG-
BOX v2 Monte Carlo (MC) event generator [19–23] for the
gluon fusion (ggH) and vector-boson fusion (VBF) proc-
esses calculated up to next-to-leading order in αS with CT10
parton distribution functions [24]. Additional contributions
from the associated production of a Higgs boson and a W or
Z boson (denoted WH and ZH, respectively) are modeled
by the PYTHIA 8.186 MC event generator [25,26] with
NNPDF 2.3 parton distribution functions [27]. The pro-
duction rates and dynamics for a SM Higgs boson with
mH ¼ 125 GeV, obtained from Ref. [28], are assumed
throughout this analysis. The ggH signal model is appro-
priately scaled to account for the production of a Higgs
boson in association with a t¯t or b¯b pair. The POWHEG-BOX
v2 MC event generator, with the CTEQ6L1 parton distri-
bution functions [29], is used to model Z boson production. The total cross section is obtained from the measurement in
Ref. [30], with an uncertainty of 5.5%. The corresponding decay of the Z boson has also been
considered from a theoretical perspective [16,17], as it
offers a precision test of the SM and the predictions of the
factorization approach in quantum chromodynamics [17]. Owing to the large Z boson production cross section at the
LHC, rare Z boson decays can be probed at branching The Higgs and Z boson decays are simulated as a
cascade of two-body decays. Effects of the helicity of
the ϕ mesons on the K kinematics are found to modify the
acceptance by at most 1% and this is corrected for in the
Higgs boson case and treated as a systematic uncertainty in
the Z boson case, due to the unknown Z boson polarization. PYTHIA 8.186 [25,26] with the AZNLO set of hadro-
nization and underlying-event parameters [31] is used to
simulate showering and hadronization. The simulated The Higgs and Z boson decays are simulated as a
cascade of two-body decays. Effects of the helicity of
the ϕ mesons on the K kinematics are found to modify the
acceptance by at most 1% and this is corrected for in the
Higgs boson case and treated as a systematic uncertainty in
the Z boson case, due to the unknown Z boson polarization. PYTHIA 8.186 [25,26] with the AZNLO set of hadro-
nization and underlying-event parameters [31] is used to
simulate showering and hadronization. *Full author list given at the end of the article.
Published by the American Physical Society under the terms of
the Creative Commons Attribution 3.0 License. Further distri-
bution of this work must maintain attribution to the author(s) and
the published article’s title, journal citation, and DOI. Search for Higgs and Z Boson Decays to ϕγ with the ATLAS Detector M. Aaboud et al.*
(ATLAS Collaboration)
(Received 14 July 2016; published 9 September 2016) A search for the decays of the Higgs and Z bosons to a ϕ meson and a photon is performed with a pp
collision data sample corresponding to an integrated luminosity of 2.7 fb−1 collected at
ffiffiffis
p ¼ 13 TeV with
the ATLAS detector at the LHC. No significant excess of events is observed above the background, and
95% confidence level upper limits on the branching fractions of the Higgs and Z boson decays to ϕγ of
1.4 × 10−3 and 8.3 × 10−6, respectively, are obtained. DOI: 10.1103/PhysRevLett.117.111802 DOI: 10.1103/PhysRevLett.117.111802 The
total signal efficiency (kinematic acceptance, and trigger
and reconstruction efficiencies) is 18% and 8% for the
Higgs and Z boson decays, respectively. The difference in
efficiencies primarily arises due to the softer pγ
T and pKþK−
T
distributions in the case of Z →ϕγ production. The mKþK−γ
resolution is around 1.8% for both the Higgs and Z boson
decays. The mKþK−distribution for selected ϕγ candidates,
with no mKþK−requirement applied, is shown in Fig. 1 and
exhibits a clear peak at the ϕ meson mass. For this analysis, in the absence of particle identification
capabilities in the relevant momentum range, every recon-
structed charged particle satisfying the following require-
ments is assumed to be a K meson. Events are selected if
there are at least two tracks with pT > 400 MeV originat-
ing from the primary vertex, which is defined as the vertex
with the largest P p2
T in the event. The charged kaons
are reconstructed from inner-detector tracks that satisfy
quality requirements, including a requirement on the
number of hits in the silicon detectors [35]. The K
candidates are required to have pseudorapidity [36] jηj <
2.5 and pT > 15 GeV. The ϕ →KþK−decays are recon-
structed from pairs of oppositely charged inner detector
tracks. The higher-pT track in a pair, denoted the leading
track, is required to have pT > 20 GeV. The experimental
resolution in mKþK−is around 4 MeV, comparable to the
natural width of the ϕ meson, Γϕ ¼ 4.266 0.031 MeV
[34]. Track pairs with a mass mKþK−within 20 MeV of
the ϕ meson mass [34] are selected as ϕ →KþK−
candidates. Selected ϕ →KþK−candidates are required
to satisfy an isolation requirement: the sum of the pT of the
reconstructed inner detector tracks from the main vertex
within ΔR ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðΔϕÞ2 þ ðΔηÞ2
p
¼ 0.2 of the leading track
(excluding both tracks constituting the ϕ →KþK−candi-
date) is required to be less than 10% of the pT of the ϕ
candidate, pKþK−
T
. [GeV]
-
K
+
K
m
0.99
1.00
1.01
1.02
1.03
1.04
1.05
Candidates / 0.002 GeV
0
20
40
60
80
100
120
140
Data
Fit Result
-
K
+
K
→
φ
Combinatoric Background
ATLAS
-1
= 13 TeV, 2.7 fb
s
FIG. 1. DOI: 10.1103/PhysRevLett.117.111802 The simulated 111802-1 © 2016 CERN, for the ATLAS Collaboration 0031-9007=16=117(11)=111802(19) week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) multiple pp interactions per bunch crossing (pile-up) in this
calculation are reduced by removing tracks that do not
originate from the primary vertex. Additionally, the sum of
the transverse momenta of all energy deposits in the
calorimeters within ΔR ¼ 0.4 of the photon direction,
excluding those associated with the reconstructed photon,
is required to be less than ð2.45 GeV þ 0.022 × pγ
TÞ. The
calorimeter isolation measurements are also corrected for
the effects of pile-up. events are passed through the detailed GEANT4 simulation
of the ATLAS detector [32,33] and processed with the same
software used to reconstruct data. events are passed through the detailed GEANT4 simulation
of the ATLAS detector [32,33] and processed with the same
software used to reconstruct data. The data sample used in this analysis was collected with
a dedicated trigger, commissioned in September 2015,
requiring an isolated photon with a transverse momentum
pT greater than 35 GeV and an isolated pair of tracks with
an invariant mass loosely consistent with the ϕ meson mass
of 1019.5 MeV [34], one of which must have a transverse
momentum greater than 15 GeV. The trigger efficiency for
both the Higgs and Z boson signals is around 80% with
respect to the offline selection. Events are retained for
analysis if collected under stable LHC beam conditions and
the detector was operating normally. Combinations of a ϕ →KþK−candidate and a photon,
satisfying ΔϕðKþK−; γÞ > 0.5, are retained for further
analysis. When multiple combinations are possible, the
combination of the highest-pT photon and the ϕ →KþK−
candidate with a mass closest to the ϕ meson mass is
retained. The transverse momentum of ϕ →KþK−candi-
dates is required to be greater than a threshold that varies as
a function of the invariant mass of the three-body system,
mKþK−γ. Thresholds of 40 GeVand 45 GeVare imposed for
the regions mKþK−γ < 91 GeV and mKþK−γ ≥125 GeV,
respectively. The threshold is varied from 40 GeV to
45 GeV as a linear function of mKþK−γ in the region
91 ≤mKþK−γ < 125 GeV. This approach ensures optimal
sensitivity for both the Higgs and Z boson searches. PRL 117, 111802 (2016) Probability density
functions (pdfs) that model the pKþK−
T
, pγ
T, ΔηðKþK−; γÞ,
and ΔϕðKþK−; γÞ distributions of this sample are con-
structed using a Gaussian kernel density estimation [40]. Correlations between these variables and pγ
T
in the
event were studied and accounted for in the background
model by deriving separate pdfs in 13 exclusive regions of
pγ
T. In the case of the ϕ →KþK−and photon isolation
variables, correlations are accounted for by using two-
dimensional histograms derived in the same 13 exclusive
regions of pγ
T. Values of mKþK−are sampled from the
corresponding distribution in the GR. The pdfs of these
kinematic and isolation variables are sampled to generate
an ensemble of pseudocandidates, each with a complete
KþK−γ four-vector and an associated pair of ϕ →KþK−
and photon isolation values. The nominal selection require-
ments are imposed on the ensemble and the surviving
pseudocandidates are used to construct templates for the
mKþK−γ distribution. FIG. 2. The distribution of mKþK−γ in data compared to the
prediction of the background model for a validation control
sample defined by the GR selection with the addition of the
nominal photon isolation requirement. The background model is
normalized to the observed number of events within the region
shown. The uncertainty band corresponds to the uncertainty
envelope derived from variations in the background modeling
procedure. threshold structures, and confirming that the background
model describes the shapes of both mKþK−γ distributions. Further exclusive background contributions from Z →llγ
decays have been studied but are found to represent a
negligible contribution for the selection requirements and
data set used in this analysis. Trigger and identification efficiencies for photons are
determined from samples enriched with Z →eþe−events
in data [37,41]. The systematic uncertainty on the expected
signal yield associated with the trigger efficiency is
estimated to be 2%. The photon identification efficiency
uncertainties, for both the converted and unconverted
photons, are estimated to be 2.4% and 2.6% for the
Higgs and Z boson signals, respectively. An uncertainty
of 6% is assigned to the track reconstruction efficiency and
includes effects associated with the material budget of the
inner detector and the behavior of the track reconstruction
algorithm if a nearby track is present. The integrated
luminosity of the data sample has an uncertainty of 5%
derived using the method described in Ref. [42]. PRL 117, 111802 (2016) PRL 117, 111802 (2016) [GeV]
γ
-
K
+
K
m
0
50
100
150
200
250
300
Events / 5 GeV
0
20
40
60
80
100
120
140
160
180
200
-1
= 13 TeV, 2.7 fb
s
Data
Background Model
Model Shape Uncertainty
ATLAS
FIG. 2. The distribution of mKþK−γ in data compared to the
prediction of the background model for a validation control
sample defined by the GR selection with the addition of the
nominal photon isolation requirement. The background model is
normalized to the observed number of events within the region
shown. The uncertainty band corresponds to the uncertainty
envelope derived from variations in the background modeling
procedure. 9 SEPTEMBER 2016 [GeV]
γ
-
K
+
K
m
0
50
100
150
200
250
300
Events / 5 GeV
0
20
40
60
80
100
120
140
160
180
200
-1
= 13 TeV, 2.7 fb
s
Data
Background Model
Model Shape Uncertainty
ATLAS The main source of background to the search comes
from events involving inclusive multijet or photon þ jet
processes where a ϕ →KþK−candidate is reconstructed
from tracks associated with a jet. The normalization of this
inclusive background is extracted directly from a fit to data. from events involving inclusive multijet or photon þ jet
processes where a ϕ →KþK−candidate is reconstructed
from tracks associated with a jet. The normalization of this
inclusive background is extracted directly from a fit to data. The selection criteria discussed earlier shape the mKþK−γ
distribution for background such that it exhibits a threshold
structure near 100 GeV, and falls then smoothly towards
higher mass values. Given the nontrivial shape of this
background, these processes are modeled with a nonpara-
metric data-driven approach using templates to describe the
kinematic distributions. A similar procedure was used in the
search for Higgs and Z boson decays to J=ψγ and ϒðnSÞγ
described in Ref. [14]. The approach exploits a sample of
around 4000 KþK−γ candidate events passing all of the
kinematic selection requirements described previously,
except that the photon and ϕ→KþK−candidates are not
required to satisfy the nominal isolation requirements. The
events satisfying this selection are collected in a generation
region (GR). The contamination of this sample from signal
events is expected to be negligible and is verified not to affect
the shape of the background model. DOI: 10.1103/PhysRevLett.117.111802 The mKþK−distribution of selected ϕγ combinations
with the complete event selection applied (see text), apart from
the requirement on mKþK−. The data are fitted with the con-
volution of a Breit-Wigner distribution, using the ϕ width [34],
and a Gaussian distribution to represent the experimental
resolution, while the background is modeled with an analytical
function, commonly used to describe a kinematic threshold [39]. [GeV]
-
K
+
K
m
0.99
1.00
1.01
1.02
1.03
1.04
1.05
Candidates / 0.002 GeV
0
20
40
60
80
100
120
140
Data
Fit Result
-
K
+
K
→
φ
Combinatoric Background
ATLAS
-1
= 13 TeV, 2.7 fb
s Photons are reconstructed from clusters of energy in the
electromagnetic calorimeter. Clusters without matching
tracks are classified as unconverted photon candidates while
clusters matched to tracks consistent with the hypothesis
of a photon conversion into an eþe−pair are classified as
converted photon candidates [37]. Reconstructed photon
candidates are required to have transverse momentum
pγ
T > 35 GeV, pseudorapidity jηγj < 2.37, excluding the
barrel or endcap calorimeter transition region 1.37 < jηγj <
1.52, and to satisfy the “tight” photon identification criteria
[38].Anisolation requirement isimposedtofurther suppress
the contamination from jets. The sum of the transverse
momenta of all tracks within ΔR ¼ 0.2 of the photon
direction, excluding those associated with the reconstructed
photon, is required to be less than 5% of pγ
T. The effects of FIG. 1. The mKþK−distribution of selected ϕγ combinations
with the complete event selection applied (see text), apart from
the requirement on mKþK−. The data are fitted with the con-
volution of a Breit-Wigner distribution, using the ϕ width [34],
and a Gaussian distribution to represent the experimental
resolution, while the background is modeled with an analytical
function, commonly used to describe a kinematic threshold [39]. FIG. 1. The mKþK−distribution of selected ϕγ combinations
with the complete event selection applied (see text), apart from
the requirement on mKþK−. The data are fitted with the con-
volution of a Breit-Wigner distribution, using the ϕ width [34],
and a Gaussian distribution to represent the experimental
resolution, while the background is modeled with an analytical
function, commonly used to describe a kinematic threshold [39]. 111802-2 111802-2 week ending
9 SEPTEMBER 2016 P H Y S I C A L
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L E T T E R S PRL 117, 111802 (2016) Events/ 5 GeV
20
40
60
80
100
120
ATLAS
-1
=13 TeV, 2.7 fb
s
Data
σ
1
±
Background Fit
Background
-3
)=10
→
B(H
-6
)=10
γ
φ
γ
φ
→
B(Z
[GeV]
γ
-
K
+
K
m
0
50
100
150
200
250
300
Data/Fit
0
0.5
1
1.5
2 300 TABLE II. Expected and observed branching fraction limits at
95% C.L. for 2.7 fb−1 of pp collision data at
ffiffiffis
p ¼ 13 TeV. The
1σ intervals of the expected limits are also given. Branching fraction limit (95% C.L.)
Expected
Observed
BðH →ϕγÞ½10−3
1.5þ0.7
−0.4
1.4
BðZ →ϕγÞ½10−6
4.4þ2.0
−1.2
8.3 TABLE II. Expected and observed branching fraction limits at
95% C.L. for 2.7 fb−1 of pp collision data at
ffiffiffis
p ¼ 13 TeV. The
1σ intervals of the expected limits are also given. TABLE II. Expected and observed branching fraction limits at
95% C.L. for 2.7 fb−1 of pp collision data at
ffiffiffis
p ¼ 13 TeV. The
1σ intervals of the expected limits are also given. Branching fraction limit (95% C.L.)
Expected
Observed
BðH →ϕγÞ½10−3
1.5þ0.7
−0.4
1.4
BðZ →ϕγÞ½10−6
4.4þ2.0
−1.2
8.3 Events/ 5 GeV On the basis of the observed data, upper limits are set on
the branching fractions for the Higgs and Z boson decays to
ϕγ using the CLs modified frequentist formalism [44] with
the profile-likelihood ratio test statistic [45]. The result of
the background-only fit is shown in Fig. 3; a small excess of
two standard deviations is observed in the Z boson mass
region, estimated using the asymptotic approximation for
the distribution of the test statistic. The expected SM
production cross section is assumed for the Higgs boson
while the ATLAS measurement of the inclusive Z boson
cross section is used for the Z boson signal [30]. The results
are summarized in Table II. The observed 95% confidence
level (C.L.) upper limits on the branching fractions for
H →ϕγ and Z →ϕγ decays are around 600 and 700 times
the expected SM branching fractions, respectively. FIG. 3. The mKþK−γ distributions of the selected ϕγ candidates,
along with the results of the maximum-likelihood fit with
background-only model. The 1σ uncertainty band corresponds
to the total uncertainty of the background model. The Higgs and
Z boson contributions, expected for branching fraction values of
10−3 and 10−6, respectively, are also shown. PRL 117, 111802 (2016) In conclusion, a search for the decay of Higgs or Z
bosons to ϕγ has been performed with a pp collision data
sample at
ffiffiffis
p ¼ 13 TeV corresponding to an integrated
luminosity of 2.7 fb−1 collected with the ATLAS detector
at the LHC. No significant excess of events is observed
above the background. Upper limits at the 95% C.L. are set
on the branching fractions for the decay of the 125 GeV SM
Higgs boson and the Z boson to ϕγ. The obtained limits are
BðH →ϕγÞ < 1.4 × 10−3 and BðZ →ϕγÞ < 8.3 × 10−6. photon energy scale uncertainty, determined from Z →
eþe−events and validated using Z →llγ events [43], is
propagated through the simulated signal samples as a
function of ηγ and pγ
T. The uncertainty associated with
the description of the photon energy scale in the simulation
is found to be less than 0.3% of the three-body invariant
mass while the uncertainty associated with the photon
energy resolution is found to be negligible relative to the
overall three-body invariant mass resolution. Similarly, the
systematic uncertainty associated with the track momentum
measurement is found to be negligible. We thank CERN for the very successful operation of the
LHC, as well as the support staff from our institutions
without whom ATLAS could not be operated efficiently. We acknowledge the support of ANPCyT, Argentina;
YerPhI, Armenia; ARC, Australia; BMWFW and FWF,
Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and
FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN;
CONICYT, Chile; CAS, MOST and NSFC, China;
COLCIENCIAS,
Colombia;
MSMT
CR,
MPO
CR
(Ministry of Industry and Trade) and VSC CR, Czech
Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS,
CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, HGF
(Helmholtz Association), and MPG, Germany; GSRT,
Greece; RGC, Hong Kong SAR, China; ISF, I-CORE
and Benoziyo Center, Israel; INFN, Italy; MEXT and
JSPS,
Japan;
CNRST,
Morocco;
FOM
and
NWO,
Netherlands; RCN, Norway; MNiSW and NCN, Poland;
FCT, Portugal; MNE/IFA, Romania; MES of Russia and
NRC KI, Russian Federation; JINR; MESTD (Ministry of
Education, Science and Technological Development),
Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia;
DST/NRF, South Africa; MINECO, Spain; SRC and The uncertainty on the shape of the inclusive multijet and
photon þ jet background is estimated through the study of
variations in the background modeling procedure. PRL 117, 111802 (2016) The γ
To validate this background model with data, the mKþK−γ
distributions in several validation regions, defined by kin-
ematic and isolation requirements looser than the nominal
signal requirements, are used to compare the prediction of
the background model with the data. The mKþK−γ distribu-
tion in one of these validation regions, defined by the GR
selection with the addition of the nominal photon isolation
requirement, is shown in Fig. 2. The background model is
found to describe the data well, and within the observed
statistical uncertainties. A consistency test of the background
modeling procedure has been performed with a sample of
simulated photon þ jet events in place of the data; similarly
good agreement is observed. The robustness of the back-
ground model is further validated by splitting the data into
high- and low-pKþK−γ
T
subsets,
that
exhibit
different TABLE I. The number of observed events and the expected
background yield for the two mKþK−γ ranges of interest. The
Higgs and Z boson contributions expected for branching fraction
values of 10−3 and 10−6, respectively, and estimated using
Monte Carlo simulations are also shown. Observed (expected) background
Expected signal
Mass range [GeV]
Z
H
All
81–101
120–130
B½10−6
B½10−3
1065
288
(266 9)
89
(87 3)
6.7 0.7 13.5 1.5 111802-3 111802-3 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) Events/ 5 GeV
20
40
60
80
100
120
ATLAS
-1
=13 TeV, 2.7 fb
s
Data
σ
1
±
Background Fit
Background
-3
)=10
→
B(H
-6
)=10
γ
φ
γ
φ
→
B(Z
[GeV]
γ
-
K
+
K
m
0
50
100
150
200
250
300
Data/Fit
0
0.5
1
1.5
2
FIG. 3. The mKþK−γ distributions of the selected ϕγ candidates,
along with the results of the maximum-likelihood fit with
background-only model. The 1σ uncertainty band corresponds
to the total uncertainty of the background model. The Higgs and
Z boson contributions, expected for branching fraction values of
10−3 and 10−6, respectively, are also shown. PRL 117, 111802 (2016) [14] ATLAS Collaboration, Search for Higgs and Z Boson
Decays to J=ψγ and ϒðnSÞγ with the ATLAS Detector,
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shape of the background model is allowed to vary around
the nominal shape within an envelope associated with
shifts in the pKþK−
T
distribution, tilts of the ΔϕðKþK−; γÞ
distribution, and by neglecting the weakest correlation
accounted for in the nominal background model. Results are compared to background and signal predic-
tions using an unbinned maximum-likelihood fit to the
mKþK−γ distribution. The fit uses the selected events with
mKþK−γ < 300 GeV.Thesystematicuncertaintiesdescribed
above result in a 3% deterioration of the sensitivity to the
H →ϕγ decay.Forthe Z bosondecay the reduction islarger,
13%, mainly due to the systematic uncertainty in the back-
ground shape. The expected and observed numbers of
background events within the mKþK−γ ranges relevant to
the Higgs and Z boson signals are shown in Table I. 111802-4 week ending
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L E T T E R S PRL 117, 111802 (2016) [12] G. T. Bodwin, F. Petriello, S. Stoynev, and M. Velasco,
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of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK,
Turkey; STFC, United Kingdom; DOE and NSF, United
States of America. In addition, individual groups and
members
have
received
support
from
BCKDF,
the
Canada Council, CANARIE, CRC, Compute Canada,
FQRNT, and the Ontario Innovation Trust, Canada;
EPLANET,
ERC,
FP7,
Horizon
2020
and
Marie
Skłodowska-Curie
Actions,
European
Union;
Investissements d’Avenir Labex and Idex, ANR, Région
Auvergne and Fondation Partager le Savoir, France; DFG
and AvH Foundation, Germany; Herakleitos, Thales and
Aristeia programmes co-financed by EU-ESF and the
Greek NSRF; BSF, GIF and Minerva, Israel; BRF
(Bergen Research Foundation), Norway; Generalitat de
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Valenciana,
Spain;
the
Royal
Society and Leverhulme Trust, United Kingdom. The
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GridKA
(Germany),
INFN-CNAF
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19
11
59b
32
75
33
92a 92b
59 gg
g
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,
,
,
,
,
,
,
,
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50
28b
32
104a l
16
79
120 Z. H. Citron,171 M. Citterio,92a M. Ciubancan,28b A. Clark,51 B. L. Clark,58 M. R. Clark,37 P. J. Clark,48 R. N. Clarke,16
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R. N. Clarke,
C. Clement,146a,146b Y. Coadou,86 M. Cobal,163a,163c A. Coccaro,51 J. Cochran,65 L. Colasurdo,106 B. Cole,37 A. P. Colijn,107
J. Collot,57 T. Colombo,32 G. Compostella,101 P. Conde Muiño,126a,126b E. Coniavitis,50 S. H. Connell,145b I. A. Connelly,78
V. Consorti,50 S. Constantinescu,28b G. Conti,32 F. Conventi,104a,l M. Cooke,16 B. D. Cooper,79 A. M. Cooper-Sarkar,120
K. J. R. Cormier,158 T. Cornelissen,174 M. Corradi,132a,132b F. Corriveau,88,m A. Corso-Radu,162 A. Cortes-Gonzalez,32
G. Cortiana,101 G. Costa,92a M. J. Costa,166 D. Costanzo,139 G. Cottin,30 G. Cowan,78 B. E. Cox,85 K. Cranmer,110
S. J. Crawley,55 G. Cree,31 S. Crépé-Renaudin,57 F. Crescioli,81 W. A. Cribbs,146a,146b M. Crispin Ortuzar,120 111802-7 P H Y S I C A L
R E V I E W
L E T T E R S Bessid ,
,
,
,
j
,
y
,
M. Bessner,44 N. Besson,136 C. Betancourt,50 A. Bethani,57 S. Bethke,101 A. J. Bevan,77 R. M. Bianchi,125 L. Bianchini,25
32
100
17
85
124 124b
134
51 M. Bianco,32 O. Biebel,100 D. Biedermann,17 R. Bielski,85 N. V. Biesuz,124a,124b M. Biglietti,134a ,
,
,
,
,
g
,
,
T. R. V. Billoud,95 H. Bilokon,49 M. Bindi,56 S. Binet,117 A. Bingul,20b C. Bini,132a,132b S. Biondi,22a,22b T. Bisanz,56 D. M. Bjergaard,47 C. W. Black,150 J. E. Black,143 K. M. Black,24 D. Blackburn,138 R. E. Blair,6 J.-B. Blanchard,136
T. Blazek,144a I. Bloch,44 C. Blocker,25 W. Blum,84,a U. Blumenschein,56 S. Blunier,34a G. J. Bobbink,107 V. S. Bobrovnikov,109,d S. S. Bocchetta,82 A. Bocci,47 C. Bock,100 M. Boehler,50 D. Boerner,174 J. A. Bogaerts,32 V. S. Bobrovnikov,109,d S. S. Bocchetta,82 A. Bocci,47 C. Bock,100 M. Boehler,50 D. Boerner,174 J. A. Bogaerts,32
D Bogavac 14 A G Bogdanchikov 109 C Bohm 146a V Boisvert 78 P Bokan 14 T Bold 40a A S Boldyrev 163a,163c D. Bogavac,14 A. G. Bogdanchikov,109 C. Bohm,146a V. Boisvert,78 P. Bokan,14 T. Bold,40a A. S. Boldyrev,163a,163c
M. Bomben,81 M. Bona,77 M. Boonekamp,136 A. Borisov,130 G. Borissov,73 J. Bortfeldt,32 D. Bortoletto,120 D. Bogavac,
A. G. Bogdanchikov,
C. Bohm,
V. Boisvert,
P. Bokan,
T. Bold,
A. S. Boldyrev,
M. Bomben,81 M. Bona,77 M. Boonekamp,136 A. Borisov,130 G. Borissov,73 J. Bortfeldt,32 D. Bortoletto,120
61a 61b 61c
107
22a
13
29
125
2 E. V. Bouhova-Thacker,73 D. Boumediene,36 C. Bourdarios,117 S. K. Boutle,55 A. Boveia,32
5
5
5 E. V. Bouhova-Thacker,73 D. Boumediene,36 C. Bourdarios,117 S. K. Boutle,55 A. Boveia,32 J. Boyd,32 I. R. Boyko,66
J B
i ik 19 A B
d 8 G B
d 56 O B
d 59a U B
l
156 B B
87 J E B
116 H M B
174 a J. Bracinik,19 A. Brandt,8 G. Brandt,56 O. Brandt,59a U. Bratzler,156 B. Brau,87 J. E. Bra W. D. Breaden Madden,55 K. Brendlinger,122 A. J. Brennan,89 L. Brenner,107 R. Brenner,164 S. Bressler,171 T. M. Bristow,48 D. Britton,
D. Britzger,
F. M. Brochu,
I. Brock,
R. Brock,
G. Brooijmans,
T. Brooks,
W. K. Brooks,
J. Brosamer,16 E. Brost,108 J. H Broughton,19 P. A. Bruckman de Renstrom,41 D. Bruncko,144b R. Bruneliere,50 A. Bruni,22a
22
107
30
22
23
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82
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J. Brosamer,16 E. Brost,108 J. H Broughton,19 P. A. Bruckman de Renstrom,41 D. P H Y S I C A L
R E V I E W
L E T T E R S Cerda Alberich,166 B. C. Cerio,47 A. S. Cerqueira,26b A. Cerri,149 L. Cerrito,133a,133b F. Cerutti,16 M. Cerv,32 A. Cervelli,18
S. A. Cetin,20c A. Chafaq,135a D. Chakraborty,108 S. K. Chan,58 Y. L. Chan,61a P. Chang,165 J. D. Chapman,30
D G Charlton 19 A Chatterjee 51 C C Chau 158 C A Chavez Barajas 149 S Che 111 S Cheatham 73 A Chegwidden 91 S. A. Cetin,20c A. Chafaq,135a D. Chakraborty,108 S. K. Chan,58 Y. L. Chan,61a P. Chang,165 J. D. Chapman,30
D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91
S Chekanov 6 S V Chekulaev 159a G A Chelkov 66,k M A Chelstowska 90 C Chen 65 H Chen 27 K Chen 148 S Chen 35c D. G. Charlton,19 A. Chatterjee,51 C. C. Chau,158 C. A. Chavez Barajas,149 S. Che,111 S. Cheatham,73 A. Chegwidden,91
S Chekanov 6 S V Chekulaev 159a G A Chelkov 66,k M A Chelstowska 90 C Chen 65 H Chen 27 K Chen 148 S Chen 35c S. Chen,155 X. Chen,35f Y. Chen,68 H. C. Cheng,90 H. J Cheng,35a Y. Cheng,33 A. Cheplakov,66 E. Cheremushkina,130
135
27
7
136
49
124 124b
74 S. Chen,155 X. Chen,35f Y. Chen,68 H. C. Cheng,90 H. J Cheng,35a Y. Cheng,33 A. Cheplakov,66 E. Cheremushkina,130
R. Cherkaoui El Moursli,135e V. Chernyatin,27,a E. Cheu,7 L. Chevalier,136 V. Chiarella,49 G. Chiarelli,124a,124b G. Chiodini,74a R. Cherkaoui El Moursli,
V. Chernyatin,
E. Cheu, L. Chevalier,
V. Chiarella,
G. Chiarelli,
G. Chiodini,
A. S. Chisholm,19 A. Chitan,28b M. V. Chizhov,66 K. Choi,62 A. R. Chomont,36 S. Chouridou,9 B. K. B. Chow,100
V. Christodoulou,79 D. Chromek-Burckhart,32 J. Chudoba,127 A. J. Chuinard,88 J. J. Chwastowski,41 L. Chytka,115
G. Ciapetti,132a,132b A. K. Ciftci,4a D. Cinca,45 V. Cindro,76 I. A. Cioara,23 C. Ciocca,22a,22b A. Ciocio,16 F. Cirotto,104a,104b A. S. Chisholm,19 A. Chitan,28b M. V. Chizhov,66 K. Choi,62 A. R. Chomont,36 S. Chouridou,9 B. K. B. Chow,100
V. Christodoulou,79 D. Chromek-Burckhart,32 J. Chudoba,127 A. J. Chuinard,88 J. J. Chwastowski,41 L. Chytka,115 Z. H. Citron,171 M. Citterio,92a M. Ciubancan,28b A. Clark,51 B. L. Clark,58 M. R. Clark,37 P. J. Clark,48 R. N. Clarke,16
C. Clement,146a,146b Y. Coadou,86 M. Cobal,163a,163c A. Coccaro,51 J. Cochran,65 L. Colasurdo,106 B. Cole,37 A. P. Colijn,107
J. Collot,57 T. Colombo,32 G. Compostella,101 P. Conde Muiño,126a,126b E. Coniavitis,50 S. H. Connell,145b I. A. Connelly,78
V. Consorti,50 S. Constantinescu,28b G. Conti,32 F. Conventi,104a,l M. Cooke,16 B. D. Cooper,79 A. M. P H Y S I C A L
R E V I E W
L E T T E R S Bruncko,144b R. Bruneliere,50 A. Bruni,22a
G B
i 22a L S B
i 107 BH B
t 30 M B
hi 22a N B
i
23 P B
t 33 L B
k 82 T B
15 G. Bruni,
L. S. Bruni,
BH Brunt,
M. Bruschi,
N. Bruscino,
P. Bryant,
L. Bryngemark,
T. Buanes,
Q. Buat,142 P. Buchholz,141 A. G. Buckley,55 I. A. Budagov,66 F. Buehrer,50 M. K. Bugge,119 O. Bulekov,98 D. Bullock,8 Q. Buat,142 P. Buchholz,141 A. G. Buckley,55 I. A. Budagov,66 F. Buehrer,50 M. K. Bugge,119 O. Bulekov,98 D. Bullock,8
H B
kh
32 S B
di
75 C D B
d 50 B B
h
108 K B
k
41 S B
k
131 I B
i
45 J T P B
120 E. Busato,36 D. Büscher,50 V. Büscher,84 P. Bussey,55 J. M. Butler,24 C. M. Buttar,55 J. M. Butterworth,79 P. Butti,107 W. Buttinger,27 A. Buzatu,55 A. R. Buzykaev,109,d S. Cabrera Urbán,166 D. Caforio,128 V. M. Cairo,39a,39b O. Cakir,4a
N. Calace,51 P. Calafiura,16 A. Calandri,86 G. Calderini,81 P. Calfayan,100 G. Callea,39a,39b L. P. Caloba,26a S. Calvente Lopez,83 D. Calvet,36 S. Calvet,36 T. P. Calvet,86 R. Camacho Toro,33 S. Camarda,32 P. Camarri,133a,133b
D. Cameron,119 R. Caminal Armadans,165 C. Camincher,57 S. Campana,32 M. Campanelli,79 A. Camplani,92a,92b
A. Campoverde,141 V. Canale,104a,104b A. Canepa,159a M. Cano Bret,35e J. Cantero,114 R. Cantrill,126a T. Cao,42 M. D. M. Capeans Garrido,32 I. Caprini,28b M. Caprini,28b M. Capua,39a,39b R. Caputo,84 R. M. Carbone,37 R. Cardarelli,133a
F. Cardillo,50 I. Carli,129 T. Carli,32 G. Carlino,104a L. Carminati,92a,92b S. Caron,106 E. Carquin,34b G. D. Carrillo-Montoya,32
30
126 126
19
13 i
13
162
145 J. R. Carter,30 J. Carvalho,126a,126c D. Casadei,19 M. P. Casado,13,i M. Casolino,13 D. W. Casper,16 J. R. Carter,30 J. Carvalho,126a,126c D. Casadei,19 M. P. Casado,13,i M. Casolino,13 D. W. Casper,162 E. Castaneda-Miranda,145a
107
107
166
126 j
32
119
32 J. R. Carter,30 J. Carvalho,126a,126c D. Casadei,19 M. P. Casado,13,i M. Casolino,13 D. W. Casper,162 E. Castaneda-Miranda,145a
R. Castelijn,107 A. Castelli,107 V. Castillo Gimenez,166 N. F. Castro,126a,j A. Catinaccio,32 J. R. Catmore,119 A. Cattai,32 R. Castelijn,107 A. Castelli,107 V. Castillo Gimenez,166 N. F. Castro,126a,j A. Catinaccio,32 J. R R. Castelijn,
A. Castelli,
V. Castillo Gimenez,
N. F. Castro,
A. Catinaccio,
J. R. Catmore,
A. Cattai,
J. Caudron,23 V. Cavaliere,165 E. Cavallaro,13 D. Cavalli,92a M. Cavalli-Sforza,13 V. Cavasinni,124a,124b F. Ceradini,134a,134b J. Caudron,23 V. Cavaliere,165 E. Cavallaro,13 D. Cavalli,92a M. Cavalli-Sforza,13 V. Cavasinni,124a,124b F. Ceradini,134a,134b L. P H Y S I C A L
R E V I E W
L E T T E R S Bagnaia,132a,132b
35
131
175
109 d
171
148
136
122 y
g
y
M. J. Baca,19 H. Bachacou,136 K. Bachas,74a,74b M. Backes,148 M. Backhaus,32 P. Bagiacchi,132a,132b P. Bagnaia,132a,132b
Y B i 35a J T B i
131 O K B k
175 E M B ldi
109,d P B l k 171 T B l
t i 148 F B lli 136 W K B l
122 E. Banas,41 Sw. Banerjee,172,f A. A. E. Bannoura,174 L. Barak,32 E. L. Barberio,89 D. Barberis,52a,52b M. Barbero,86 T. Barillari,101 M-S Barisits,32 T. Barklow,143 N. Barlow,30 S. L. Barnes,85 B. M. Barnett,131 R. M
5 T. Barillari,
M-S Barisits,
T. Barklow,
N. Barlow,
S. L. Barnes,
B. M. Barnett,
R. M. Barnett,
Z. Barnovska,
A. Baroncelli,134a G. Barone,25 A. J. Barr,120 L. Barranco Navarro,166 F. Barreiro,83 J. Barreiro Guimarães da Costa,35a A. Baroncelli,134a G. Barone,25 A. J. Barr,120 L. Barranco Navarro,166 F. Barreiro,83 J. Barreiro Guimarães da Costa,35a R. Bartoldus,143 A. E. Barton,73 P. Bartos,144a A. Basalaev,123 A. Bassalat,117 R. L. Bates,55 S. J. Batista,158 J. R. Batley,30
M. Battaglia,137 M. Bauce,132a,132b F. Bauer,136 H. S. Bawa,143,g J. B. Beacham,111 M. D. Beattie,73 T. Beau,81 ,
,
,
,
,
,
,
y,
M. Battaglia,137 M. Bauce,132a,132b F. Bauer,136 H. S. Bawa,143,g J. B. Beacham,111 M. D. Beattie,73 T. Beau,81 g
A. J. Beddall,20d A. Beddall,20b V. A. Bednyakov,66 M. Bedognetti,107 C. P. Bee,148 L. J. Beemster,107 T. A. Beermann,32 A. J. Beddall,20d A. Beddall,20b V. A. Bednyakov,66 M. Bedognetti,107 C. P. Bee,148 L. J. Beemster,107 T. A. Beermann,32
M. Begel,27 J. K. Behr,44 C. Belanger-Champagne,88 A. S. Bell,79 G. Bella,153 L. Bellagamba,22a A. Bellerive,31 111802-6 week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) M. Bellomo,87 K. Belotskiy,98 O. Beltramello,32 N. L. Belyaev,98 O. Benary,153 D. Benchekroun,135a M. Bender,100
K. Bendtz,146a,146b N. Benekos,10 Y. Benhammou,153 E. Benhar Noccioli,175 J. Benitez,64 D. P. Benjamin,47 J. R. Bensinger,25 S. Bentvelsen,107 L. Beresford,120 M. Beretta,49 D. Berge,107 E. Bergeaas Kuutmann,164 N. Berger,5 J. Beringer,16
S. Berlendis,57 N. R. Bernard,87 C. Bernius,110 F. U. Bernlochner,23 T. Berry,78 P. Berta,129 C. Bertella,84 G. Bertoli,146a,146b ,
,
,
,
y,
,
F. Bertolucci,124a,124b I. A. Bertram,73 C. Bertsche,44 D. Bertsche,113 G. J. Besjes,38 O. 111802-7 week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) M. Cristinziani,23 V. Croft,106 G. Crosetti,39a,39b A. Cueto,83 T. Cuhadar Donszelmann,139 J. Cummings,175 M. Curatolo,49
J. Cúth,84 H. Czirr,141 P. Czodrowski,3 G. D’amen,22a,22b S. D’Auria,55 M. D’Onofrio,75 M. Cristinziani,
V. Croft,
G. Crosetti,
A. Cueto,
T. Cuhadar Donszelmann,
J. Cummings,
M. Curatolo,
J. Cúth,84 H. Czirr,141 P. Czodrowski,3 G. D’amen,22a,22b S. D’Auria,55 M. D’Onofrio,75 M. J. Da Cunha Sargedas De Sousa,126a,126b C. Da Via,85 W. Dabrowski,40a T. Dado,144a T. Dai,90 O. Dale,15 F. Dallaire,95
C. Dallapiccola,87 M. Dam,38 J. R. Dandoy,33 N. P. Dang,50 A. C. Daniells,19 N. S. Dann,85 M. Danninger,167 ,
,
y,
g,
,
,
fmann,136 V. Dao,50 G. Darbo,52a S. Darmora,8 J. Dassoulas,3 A. Dattagupta,62 W. Davey,23 C p
y
g
M. Dano Hoffmann,136 V. Dao,50 G. Darbo,52a S. Darmora,8 J. Dassoulas,3 A. Dattagupta,62 W
129
153
79
89
139 p
y
g
g
M. Dano Hoffmann,136 V. Dao,50 G. Darbo,52a S. Darmora,8 J. Dassoulas,3 A. Dattagupta,62 W. Davey,23 C. David,168
T. Davidek,129 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. De,8 M. Dano Hoffmann,
V. Dao,
G. Darbo,
S. Darmora, J. Dassoulas, A. Dattagupta,
W. Davey,
C. David,
T. Davidek,129 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. De,8
R d A
di
104a A D B
d
i 113 S D C
22a 22b S D C
81 N D G
106 P d J
107 H D l T
83 29 M. Davies,153 P. Davison,79 E. Dawe,89 I. Dawson,139 R. K. Daya-Ishmukhametova,87 K. D y
A. De Benedetti,113 S. De Castro,22a,22b S. De Cecco,81 N. De Groot,106 P. de Jong,107 H. De la T y
de Asmundis,104a A. De Benedetti,113 S. De Castro,22a,22b S. De Cecco,81 N. De Groot,106 P. de J g
F. De Lorenzi,65 A. De Maria,56 D. De Pedis,132a A. De Salvo,132a U. De Sanctis,149 A. De Santo,149 J. B. De Vivie De Regie,117 W. J. Dearnaley,73 R. Debbe,27 C. Debenedetti,137 D. V. Dedovich,66 N. Dehghanian,3
I. Deigaard,107 M. Del Gaudio,39a,39b J. Del Peso,83 T. Del Prete,124a,124b D. Delgove,117 F. Deliot,136 C. M. Delitzsch,51 vie De Regie,117 W. J. Dearnaley,73 R. Debbe,27 C. Debenedetti,137 D. V. Dedovich,66 N. Deh g
g
A. Dell’Acqua,32 L. Dell’Asta,24 M. 111802-7 Dell’Orso,124a,124b M. Della Pietra,104a,l D. della Volpe,51 M. Delmastro,5 P. A. Delsart,57 D. A. DeMarco,158 S. Demers,175 M. Demichev,66 A. Demilly,81 S. P. Denisov,130 D. Denysiu J. E. Derkaoui,135d F. Derue,81 P. Dervan,75 K. Desch,23 C. Deterre,44 K. Dette,45 P. O. Deviveiros,32 A. Dewhurst,131 S. Dhaliwal,25 A. Di Ciaccio,133a,133b L. Di Ciaccio,5 W. K. Di Clemente,122 C. Di Donato,132a B. Di Girolamo,32 B. Di Micco,134a,134b R. Di Nardo,32 A. Di Simone,50 R. Di Sipio,158 D. Di
158
48
34
90
17
86 B. Di Girolamo,32 B. Di Micco,134a,134b R. Di Nardo,32 A. Di Simone,50 R. Di Sipio,158 D. Di Valentino,31 C. Diaconu,86
M Di
d 158 F A Di
48 M A Di
34a E B Di hl 90 J Di
i h 17 S Di li
86 A Di i i
k
14 J Di
f ld
23 M. Diamond,158 F. A. Dias,48 M. A. Diaz,34a E. B. Diehl,90 J. Dietrich,17 S. Diglio,86 A. Dim P. Dita,28b S. Dita,28b F. Dittus,32 F. Djama,86 T. Djobava,53b J. I. Djuvsland,59a M. A. B. do Vale,2 P. Dita,28b S. Dita,28b F. Dittus,32 F. Djama,86 T. Djobava,53b J. I. Djuvsland,59a M. A. B. do Vale,26c D. Dobos,32 M. Dobre,28b
82
129
129
26d
124 124b
121 121b
36
131 C. Doglioni,82 J. Dolejsi,129 Z. Dolezal,129 M. Donadelli,26d S. Donati,124a,124b P. Dondero,121a,1 A. Doria,104a M. T. Dova,72 A. T. Doyle,55 E. Drechsler,56 M. Dris,10 Y. Du,35d J. Duarte-Campderros,153 E. Duchovni,171 A. Doria,104a M. T. Dova,72 A. T. Doyle,55 E. Drechsler,56 M. Dris,10 Y. Du,35d J. Duarte-C G. Duckeck,100 O. A. Ducu,95,n D. Duda,107 A. Dudarev,32 A. Chr. Dudder,84 E. M. Duffield,16 L. Duflot,117 M. Dührssen,32
M. Dumancic,171 M. Dunford,59a H. Duran Yildiz,4a M. Düren,54 A. Durglishvili,53b D. Duschinger,46 B. Dutta,44
M. Dyndal,44 C. Eckardt,44 K. M. Ecker,101 R. C. Edgar,90 N. C. Edwards,48 T. Eifert,32 G. Eigen,15 K. Einsweiler,16 M. Dyndal,44 C. Eckardt,44 K. M. Ecker,101 R. C. Edgar,90 N. C. Edwards,48 T. Eifert,32 G. Eigen,15 K. Einsweiler,16
T. Ekelof,164 M. El Kacimi,135c V. Ellajosyula,86 M. Ellert,164 S. Elles,5 F. Ellinghaus,174 A. A. Elliot,168 N. Ellis,32 T. Ekelof,164 M. El Kacimi,135c V. Ellajosyula,86 M. Ellert,164 S. Elles,5 F. Ellinghaus,174 A. A. Elliot,168 N. Ellis,32 J. Elmsheuser,27 M. Elsing,32 D. Emeliyanov,131 Y. Enari,155 O. C. Endner,84 J. S. Ennis,169 J. Erdmann,45 A. Ereditato,18
G. Ernis,174 J. Ernst,2 M. Ernst,27 S. Errede,165 E. Ertel,84 M. Escalier,117 H. Esch,45 C. Escobar,125 B. Esposito,49 g
y
G. 111802-7 Gomes ,
,
,
g,
,
,
ç
,
J. Goncalves Pinto Firmino Da Costa,136 G. Gonella,50 L. Gonella,19 A. Gongadze,66 S. González de la Hoz,166
13
51
32
97
27
105
32 J. Goncalves Pinto Firmino Da Costa,136 G. Gonella,50 L. Gonella,19 A. Gongadze,66 S. González de la Hoz,166
G. Gonzalez Parra,13 S. Gonzalez-Sevilla,51 L. Goossens,32 P. A. Gorbounov,97 H. A. Gordon,27 I. Gorelov,105 B. Gorini,32 E. Gorini,74a,74b A. Gorišek,76 E. Gornicki,41 A. T. Goshaw,47 C. Gössling,45 M. I. Gostkin,66 C. R. Goudet,117
5
5
5
5
5 g
A. G. Goussiou,138 N. Govender,145b,q E. Gozani,152 L. Graber,56 I. Grabowska-Bold,40a P. O. J. G g
D. Goujdami,135c A. G. Goussiou,138 N. Govender,145b,q E. Gozani,152 L. Graber,56 I. Grabowsk 22b J. Gramling,51 E. Gramstad,119 S. Grancagnolo,17 V. Gratchev,123 P. M. Gravila,28e H. M. P. Grafström,22a,22b J. Gramling,51 E. Gramstad,119 S. Grancagnolo,17 V. Gratchev,123 P. M. Gravila,28e H. M. Gray,32 g,
,
g
,
,
,
D. Greenwood,80,r C. Grefe,23 K. Gregersen,79 I. M. Gregor,44 P. Grenier,143 K. Grevtsov,5 J. G Graziani,134a Z. D. Greenwood,80,r C. Grefe,23 K. Gregersen,79 I. M. Gregor,44 P. Grenier,143 K E. Graziani,134a Z. D. Greenwood,80,r C. Grefe,23 K. Gregersen,79 I. M. Gregor,44 P. Grenier,143 K. Grevtsov,5 J. Griffiths,8
A. A. Grillo,137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. Grohs,46 E. Gross,171
56
80
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90
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63
51
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A. A. Grillo,137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. Grohs,46 E. Gross,171 g
g
A. A. Grillo,137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. G g
g
137 K. Grimm,73 S. Grinstein,13,s Ph. Gris,36 J.-F. Grivaz,117 S. Groh,84 J. P. Grohs,46 E. Gross J. Grosse-Knetter,56 G. C. Grossi,80 Z. J. Grout,79 L. Guan,90 W. Guan,172 J. Guenther,63 F. Guescini,51 D. Guest,162
O Gueta 153 E Guido 52a,52b T Guillemin 5 S Guindon 2 U Gul 55 C Gumpert 32 J Guo 35e Y Guo 35b,p R Gupta 42 J. Grosse-Knetter,56 G. C. Grossi,80 Z. J. Grout,79 L. Guan,90 W. Guan,172 J. Guenther,63 F. Guescini,51 D. Guest,162 E. Guido,52a,52b T. Guillemin,5 S. Guindon,2 U. Gul,55 C. Gumpert,32 J. Guo,35e Y. Guo,35b,p R O. Gueta,153 E. Guido,52a,52b T. Guillemin,5 S. Guindon,2 U. Gul,55 C. Gumpert,32 J. Guo,35e Y. Guo,35b,p R. Gupta,42
S. Gupta,120 G. Gustavino,132a,132b P. Gutierrez,113 N. G. Gutierrez Ortiz,79 C. 111802-7 Gutschow,46 C. Guyot,136 C. Gwenlan,120
5
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S. Gupta,120 G. Gustavino,132a,132b P. Gutierrez,113 N. G. Gutierrez Ortiz,79 C. Gutschow,46 C. Guyot,136 C. Gwenlan,120
5
110
16
8
135
86
23
41 . Gustavino,132a,132b P. Gutierrez,113 N. G. Gutierrez Ortiz,79 C. Gutschow,46 C. Guyot,136 C. G C. B. Gwilliam,75 A. Haas,110 C. Haber,16 H. K. Hadavand,8 N. Haddad,135e A. Hadef,86 S. Hageböck,23 Z. Hajduk,41
H. Hakobyan,176,a M. Haleem,44 J. Haley,114 G. Halladjian,91 G. D. Hallewell,86 K. Hamacher,174 P. Hamal,115 Hamano,168 A. Hamilton,145a G. N. Hamity,139 P. G. Hamnett,44 L. Han,35b K. Hanagaki,67,t K. H Hamilton,145a G. N. Hamity,139 P. G. Hamnett,44 L. Han,35b K. Hanagaki,67,t K. Hanawa,155 M. H B. Haney,122 S. Hanisch,32 P. Hanke,59a R. Hanna,136 J. B. Hansen,38 J. D. Hansen,38 M. C. Hansen,23 P. H. Hansen,38
6
6 Hard,172 T. Harenberg,174 F. Hariri,117 S. Harkusha,93 R. D. Harrington,48 P. F. Harrison,169 F. H K. Hara,160 A. S. Hard,172 T. Harenberg,174 F. Hariri,117 S. Harkusha,93 R. D. Harrington,48 P. F N. M. Hartmann,100 M. Hasegawa,68 Y. Hasegawa,140 A. Hasib,113 S. Hassani,136 S. Haug,18 R. Hauser,91 L. Hauswald,46
127
19
32
157
91
120
77 N. M. Hartmann,100 M. Hasegawa,68 Y. Hasegawa,140 A. Hasib,113 S. Hassani,136 S. Haug,18 R. Hauser,91 L. Hauswald,46
M. Havranek,127 C. M. Hawkes,19 R. J. Hawkings,32 D. Hayakawa,157 D. Hayden,91 C. P. Hays,120 J. M. Hays,77
H S Hayward 75 S J Haywood 131 S J Head 19 T Heck 84 V Hedberg 82 L Heelan 8 S Heim 122 T Heim 16 N. M. Hartmann,100 M. Hasegawa,68 Y. Hasegawa,140 A. Hasib,113 S. Hassani,136 S. Haug,18 R. Hauser,91 L. Hauswald,46
M. Havranek,127 C. M. Hawkes,19 R. J. Hawkings,32 D. Hayakawa,157 D. Hayden,91 C. P. Hays,120 J. M. Hays,77
H. S. Hayward,75 S. J. Haywood,131 S. J. Head,19 T. Heck,84 V. Hedberg,82 L. Heelan,8 S. Heim,122 T. Heim,16 27 C. M. Hawkes,19 R. J. Hawkings,32 D. Hayakawa,157 D. Hayden,91 C. P. Hays,120 J. M. Ha ,
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H. S. Hayward,75 S. J. Haywood,131 S. J. Head,19 T. Heck,84 V. Hedberg,82 L. Heelan,8 S. Heim,122 T. Heim,16 Hayward,75 S. J. Haywood,131 S. J. Head,19 T. Heck,84 V. Hedberg,82 L. Heelan,8 S. Heim,122 y
y
g
Heinemann,16 J. J. Heinrich,100 L. Heinrich,110 C. Heinz,54 J. Hejbal,127 L. Helary,32 S. Hellm J. Henderson,120 R. C. W. 111802-7 Ernis,174 J. Ernst,2 M. Ernst,27 S. Errede,165 E. Ertel,84 M. Escalier,117 H. Esch,45 C. Escobar,125 B. Esposito,49 A. I. Etienvre,136 E. Etzion,153 H. Evans,62 A. Ezhilov,123 F. Fabbri,22a,22b L. Fabbri,22a,22b G. Facini,33 R. M. Fakhrutdinov,130
S. Falciano,132a R. J. Falla,79 J. Faltova,32 Y. Fang,35a M. Fanti,92a,92b A. Farbin,8 A. Farilla,134a C. Farina,125 A. I. Etienvre,136 E. Etzion,153 H. Evans,62 A. Ezhilov,123 F. Fabbri,22a,22b L. Fabbri,22a,22b G. Facini,33 R. M. Fakhrutdinov,130
S. Falciano,132a R. J. Falla,79 J. Faltova,32 Y. Fang,35a M. Fanti,92a,92b A. Farbin,8 A. Farilla,134a C. Farina,125
E M Farina 121a,121b T Farooque 13 S Farrell 16 S M Farrington 169 P Farthouat 32 F Fassi 135e P Fassnacht 32 D. Fassouliotis,9 M. Faucci Giannelli,78 A. Favareto,52a,52b W. J. Fawcett,120 L. Fayard,117 O. L. Fedin,123,o W. Fedorko,167 S. Feigl,119 L. Feligioni,86 C. Feng,35d E. J. Feng,32 H. Feng,90 A. B. Fenyuk,130 L. Feremenga S. Fernandez Perez,13 J. Ferrando,55 A. Ferrari,164 P. Ferrari,107 R. Ferrari,121a D. E. Ferreira de Lima,59b A. Ferrer,166
D. Ferrere,51 C. Ferretti,90 A. Ferretto Parodi,52a,52b F. Fiedler,84 A. Filipčič,76 M. Filipuzzi,44 F. Filthaut,106
M. Fincke-Keeler,168 K. D. Finelli,150 M. C. N. Fiolhais,126a,126c L. Fiorini,166 A. Firan,42 A. Fischer,2 C. Fischer,13 J. Fischer,174 W. C. Fisher,91 N. Flaschel,44 I. Fleck,141 P. Fleischmann,90 G. T. Fletcher,139 R. R A. Floderus,82 L. R. Flores Castillo,61a M. J. Flowerdew,101 G. T. Forcolin,85 A. Formica,136 D. Fournier,117 H. Fox,73 S. Fracchia,13 P. Francavilla,81 M. Franchini,22a,22b D. Francis,32 L. Franconi,119 M. Franklin,58
M. Frate,162 M. Fraternali,121a,121b D. Freeborn,79 S. M. Fressard-Batraneanu,32 F. Friedrich,46 D. Froidevaux,32
J. A. Frost,120 C. Fukunaga,156 E. Fullana Torregrosa,84 T. Fusayasu,102 J. Fuster,166 C. Gabaldon,57 O. Gabizon,174
A. Gabrielli,22a,22b A. Gabrielli,16 G. P. Gach,40a S. Gadatsch,32 S. Gadomski,51 G. Gagliardi,52a,52b L. G. Gagnon,95 P. Gagnon,62 C. Galea,106 B. Galhardo,126a,126c E. J. Gallas,120 B. J. Gallop,131 P. Gallus,128 G. Galster,38 K. K. Gan,111
35b 86
48
143
48
166
166
16 g
g
g
g
P. F. Giraud,136 P. Giromini,58 D. Giugni,92a F. Giuli,120 C. Giuliani,101 M. Giulini,59b B. K. Gjelsten,119 S. Gkaitatzis,154
I. Gkialas,154 E. L. Gkougkousis,117 L. K. Gladilin,99 C. Glasman,83 J. Glatzer,50 P. C. F. Glaysher,48 A. Glazov,44 111802-8 week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) M. Goblirsch-Kolb,25 J. Godlewski,41 S. Goldfarb,89 T. Golling,51 D. Golubkov,130 A. 111802-7 Henderson,73 Y. Heng,172 S. Henkelmann,167 A. M. Henriques Correia,32 S. Henrot-Versille,117
17
173
166
50
100
32
79 J. Henderson,120 R. C. W. Henderson,73 Y. Heng,172 S. Henkelmann,167 A. M. Henriques Correia,32 S. Henrot-Versille,117
G. H. Herbert,17 V. Herget,173 Y. Hernández Jiménez,166 G. Herten,50 R. Hertenberger,100 L. Hervas,32 G. G. Hesketh,79 N. P. Hessey,107 J. W. Hetherly,42 R. Hickling,77 E. Higón-Rodriguez,166 E. Hill,168 J. C. Hill,30 K. H. Hiller,44 S. J. Hillier,19
I. Hinchliffe,16 E. Hines,122 R. R. Hinman,16 M. Hirose,50 D. Hirschbuehl,174 J. Hobbs,148 N. Hod,159a M. C. Hodgkinson,139 N. P. Hessey,107 J. W. Hetherly,42 R. Hickling,77 E. Higón-Rodriguez,166 E. Hill,168 J. C. Hill,30 K. H. Hiller,44 S. J. Hillier,19
I Hi
hliff
16 E Hi
122 R R Hi
16 M Hi
50 D Hi
hb
hl 174 J H bb
148 N H d 159a M C H d ki
139 P. Hodgson,139 A. Hoecker,32 M. R. Hoeferkamp,105 F. Hoenig,100 D. Hohn,23 T. R. Holmes,16 M. Homann,45 T. M. Hong,125
165
116
103
142
57
151
135a P. Hodgson,139 A. Hoecker,32 M. R. Hoeferkamp,105 F. Hoenig,100 D. Hohn,23 T. R. Holmes,16 M. Homann,45 T. M. Hong,125
B. H. Hooberman,165 W. H. Hopkins,116 Y. Horii,103 A. J. Horton,142 J-Y. Hostachy,57 S. Hou,151 A. Hoummada,135a
J. Howarth,44 M. Hrabovsky,115 I. Hristova,17 J. Hrivnac,117 T. Hryn’ova,5 A. Hrynevich,94 C. Hsu,145c P. J. Hsu,151,u
S.-C. Hsu,138 D. Hu,37 Q. Hu,35b S. Hu,35e Y. Huang,44 Z. Hubacek,128 F. Hubaut,86 F. Huegging,23 T. B. Huffman,120
E. W. Hughes,37 G. Hughes,73 M. Huhtinen,32 P. Huo,148 N. Huseynov,66,c J. Huston,91 J. Huth,58 G. Iacobucci,51
G. Iakovidis,27 I. Ibragimov,141 L. Iconomidou-Fayard,117 E. Ideal,175 Z. Idrissi,135e P. Iengo,32 O. Igonkina,107,v T. Iizawa,170
Y Ik
i 67 M Ik
67 Y Il h
k
11,w D Ili di
154 N Ili
143 T I
101 G I t
i 121a,121b P I
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134a J. Howarth,44 M. Hrabovsky,115 I. Hristova,17 J. Hrivnac,117 T. Hryn’ova,5 A. Hrynevich,94 C. Hsu,145c P. J. Hsu,151,u
S.-C. Hsu,138 D. Hu,37 Q. Hu,35b S. Hu,35e Y. Huang,44 Z. Hubacek,128 F. Hubaut,86 F. Huegging,23 T. B. Huffman,120
E. W. Hughes,37 G. Hughes,73 M. Huhtinen,32 P. Huo,148 N. Huseynov,66,c J. Huston,91 J. Huth,58 G. Iacobucci,51
G. Iakovidis,27 I. Ibragimov,141 L. Iconomidou-Fayard,117 E. Ideal,175 Z. Idrissi,135e P. Iengo,32 O. Igonkina,107,v T. Iizawa,170 G. Iakovidis,
I. Ibragimov,
L. Iconomidou Fayard,
E. Ideal,
Z. Idrissi,
P. Iengo,
O. Igonkina,
T. Iizawa,
Y. Ikegami,67 M. Ikeno,67 Y. 111802-7 Ilchenko,11,w D. Iliadis,154 N. Ilic,143 T. Ince,101 G. Introzzi,121a,121b P. Ioannou,9,a M. Iodice,134a Y. Ikegami,67 M. Ikeno,67 Y. Ilchenko,11,w D. Iliadis,154 N. Ilic,143 T. Ince,101 G. Introzzi,121a,121b P. Ioannou,9,a M. Iodice,134a K. Iordanidou,37 V. Ippolito,58 N. Ishijima,118 M. Ishino,155 M. Ishitsuka,157 R. Ishmukhametov,111 C. Issever,120 S. Istin,20a
F. Ito,160 J. M. Iturbe Ponce,85 R. Iuppa,133a,133b W. Iwanski,41 H. Iwasaki,67 J. M. Izen,43 V. Izzo,104a S. Jabbar,3 S. Jiggins,79 J. Jimenez Pena,166 S. Jin,35a A. Jinaru,28b O. Jinnouchi,157 H. Jivan,145c P. Johansson,139 K. A. Johns,7
W. J. Johnson,138 K. Jon-And,146a,146b G. Jones,169 R. W. L. Jones,73 S. Jones,7 T. J. Jones,75 J. Jongmanns,59a
P. M. Jorge,126a,126b J. Jovicevic,159a X. Ju,172 A. Juste Rozas,13,s M. K. Köhler,171 A. Kaczmarska,41 M. Kado,117
H. Kagan,111 M. Kagan,143 S. J. Kahn,86 T. Kaji,170 E. Kajomovitz,47 C. W. Kalderon,120 A. Kaluza,84 S. Kama,42
130
155
30
76
98
67
110
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P. M. Jorge,126a,126b J. Jovicevic,159a X. Ju,172 A. Juste Rozas,13,s M. K. Köhler,171 A. Kaczmarska,41 M. Kado,117
H. Kagan,111 M. Kagan,143 S. J. Kahn,86 T. Kaji,170 E. Kajomovitz,47 C. W. Kalderon,120 A. Kaluza,84 S. Kama,42
130
155
30
76
98
67
110
172 A. Kamenshchikov,130 N. Kanaya,155 S. Kaneti,30 L. Kanjir,76 V. A. Kantserov,98 J. Kanzaki,67 B. Kaplan,110 L. S. Kaplan,172
A. Kapliy,33 D. Kar,145c K. Karakostas,10 A. Karamaoun,3 N. Karastathis,10 M. J. Kareem,56 E. Karentzos,10 M. Karnevskiy,84 S. N. Karpov,66 Z. M. Karpova,66 K. Karthik,110 V. Kartvelishvili,73 A. N. Ka L. Kashif,172 R. D. Kass,111 A. Kastanas,15 Y. Kataoka,155 C. Kato,155 A. Katre,51 J. Katzy,44 K. Kawagoe,71 T. Kawamoto,155
G. Kawamura,56 V. F. Kazanin,109,d R. Keeler,168 R. Kehoe,42 J. S. Keller,44 J. J. Kempster,78 K Kentaro,103
H. Keoshkerian,158 O. Kepka,127 B. P. Kerševan,76 S. Kersten,174 R. A. Keyes,88 M. Khader,165 F. Khalil-zada,12
A. Khanov,114 A. G. Kharlamov,109,d T. J. Khoo,51 V. Khovanskiy,97 E. Khramov,66 J. Khubua,53b,z S. Kido,68 C. R. Kilby,78
H. Y. Kim,8 S. H. Kim,160 Y. K. Kim,33 N. Kimura,154 O. M. Kind,17 B. T. King,75 M. King,166 S. B. King,167 J. Kirk,131
A. E. Kiryunin,101 T. Kishimoto,155 D. Kisielewska,40a F. Kiss,50 K. Kiuchi,160 O. Kivernyk,136 E. Kladiva,144b M. H. Klein,37 111802-9 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) PRL 117, 111802 (2016) M. Klein,75 U. Klein,75 K. Kleinknecht,84 P. Klimek,108 A. Klimentov,27 R. Klingenberg,45 J. A. Klinger,139
T. Klioutchnikova,32 E.-E. Kluge,59a P. Kluit,107 S. Kluth,101 J. Knapik,41 E. Kneringer,63 E. B. F. G. Knoops,86 A. Knue,55 A. Kobayashi,155 D. Kobayashi,157 T. Kobayashi,155 M. Kobel,46 M. Kocian,143 P. Kodys,129 N. M. Koehler,101 T. Koffas,31
E. Koffeman,107 T. Koi,143 H. Kolanoski,17 M. Kolb,59b I. Koletsou,5 A. A. Komar,96,a Y. Komori,155 T. Kondo,67 U. Kruchonak,
H. Krüger,
N. Krumnack,
A. Kruse,
M. C. Kruse,
M. Kruskal,
T. Kubota,
H. Kucuk,
S. Kuday,4b J. T. Kuechler,174 S. Kuehn,50 A. Kugel,59c F. Kuger,173 A. Kuhl,137 T. Kuhl,44 V. Kukhtin,66 R. Kukla,136 A. E. Loevschall-Jensen,38 K. M. Loew,25 A. Loginov,175,a T. Lohse,17 K. Lohwasser,44 M. Lokajicek,127 B. A. Long,24
J D L
165 R E L
73 L L
74a 74b K A L
111 L L
126a D L
M
58 B L
P
d
139 g
g
g
p
p
p
p
I. Lopez Paz,13 A. Lopez Solis,81 J. Lorenz,100 N. Lorenzo Martinez,62 M. Losada,21 P. J. Lösel,100 X. Lou,35a A. Lounis,117 g
W. Lukas,63 L. Luminari,132a O. Lundberg,146a,146b B. Lund-Jensen,147 P. M. Luzi,81 D. Lynn,27 R. Lysak,127 E. Lytken,82
66
27
35d
35d
49
101
139
76 y
,
,
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,
,
,
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J. Machado Miguens,122,126b D. Madaffari,86 R. Madar,36 H. J. Maddocks,164 W. F. Mader,46 A. Madsen,44 J. Maeda,68
15
27
99
56
107
117
26
101 S. Maeland,15 T. Maeno,27 A. Maevskiy,99 E. Magradze,56 J. Mahlstedt,107 C. Maiani,117 C. Maidantchik,26a A. A. Maier,101
T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41
123
57
64
6
143
10
32
166 S. Maeland,15 T. Maeno,27 A. Maevskiy,99 E. Magradze,56 J. Mahlstedt,107 C. Maiani,117 C. Maidantchik,26a A. A. Maier,101
T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41
V. P. Maleev,123 F. Malek,57 U. Mallik,64 D. Malon,6 C. Malone,143 S. Maltezos,10 S. Malyukov,32 J. Mamuzic,166
G. Mancini,49 B. Mandelli,32 L. Mandelli,92a I. Mandić,76 J. Maneira,126a,126b L. Manhaes de Andrade Filho,26b
159b
100
32
136
35
88
56 ,
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T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41
V. P. Maleev,123 F. Malek,57 U. Mallik,64 D. Malon,6 C. Malone,143 S. Maltezos,10 S. Malyukov,32 J. Mamuzic,166
G. Mancini,49 B. Mandelli,32 L. Mandelli,92a I. Mandić,76 J. Maneira,126a,126b L. PRL 117, 111802 (2016) Minashvili,66 A. M. Mineev,66 Y. Ming,172 L. M. Mir,13 K. P. Mistry,122 T. Mitani,170 J. Mitrevski,100 V. A. Mitsou,166 A. Miucci,51
P. S. Miyagawa,139 J. U. Mjörnmark,82 T. Moa,146a,146b K. Mochizuki,95 S. Mohapatra,37 S. Molander,146a,146b
R. Moles-Valls,23 R. Monden,69 M. C. Mondragon,91 K. Mönig,44 J. Monk,38 E. Monnier,86 A. Montalbano,148 Valls,23 R. Monden,69 M. C. Mondragon,91 K. Mönig,44 J. Monk,38 E. Monnier,86 A. Montalb g
g
J. Montejo Berlingen,32 F. Monticelli,72 S. Monzani,92a,92b R. W. Moore,3 N. Morange,117 D. Moreno,21 M. Moreno Llácer,56 J. Montejo Berlingen,32 F. Monticelli,72 S. Monzani,92a,92b R. W. Moore,3 N. Morange,117 D P. Morettini,52a D. Mori,142 T. Mori,155 M. Morii,58 M. Morinaga,155 V. Morisbak,119 S. M
5 b G. Mornacchi,32 J. D. Morris,77 S. S. Mortensen,38 L. Morvaj,148 M. Mosidze,53b J. Moss,143 K. Motohashi,157 R. Mount,143
5 G. Mornacchi,32 J. D. Morris,77 S. S. Mortensen,38 L. Morvaj,148 M. Mosidze,53b J. Moss,143 K. E. Mountricha,27 S. V. Mouraviev,96,a E. J. W. Moyse,87 S. Muanza,86 R. D. Mudd,19 F. Mueller,101 J. Mueller,125
R. S. P. Mueller,100 T. Mueller,30 D. Muenstermann,73 P. Mullen,55 G. A. Mullier,18 F. J. Munoz Sanchez,85 B. P. Nachman,143 O. Nackenhorst,51 K. Nagai,120 R. Nagai,67,aa K. Nagano,67 Y. Nagasaka,60 K. Nagata,160 M. Nagel,50
E. Nagy,86 A. M. Nairz,32 Y. Nakahama,103 K. Nakamura,67 T. Nakamura,155 I. Nakano,112 H. Namasivayam,43 R. F. Naranjo Garcia,44 R. Narayan,11 D. I. Narrias Villar,59a I. Naryshkin,123 T. Naumann,44 G. Navarro,21 R. Nayyar,7 S. Nemecek,127 P. Nemethy,110 A. A. Nepomuceno,26a M. Nessi,32,gg M. S. Neubauer,165 M. Neumann,174 R. M. Neves,110
2
19
6
95
120
136
13 A. Nikiforov,17 V. Nikolaenko,130,ff I. Nikolic-Audit,81 K. Nikolopoulos,19 J. K. Nilsen,119 P. Nilsson,27 Y. Ninomiya,155
A. Nisati,132a R. Nisius,101 T. Nobe,155 M. Nomachi,118 I. Nomidis,31 T. Nooney,77 S. Norberg,113 M. Nordberg,32 y
g
g
N. Norjoharuddeen,120 O. Novgorodova,46 S. Nowak,101 M. Nozaki,67 L. Nozka,115 K. Ntekas,10 E. Nurse,79 F. Nuti,89 F. O’grady,7 D. C. O’Neil,142 A. A. O’Rourke,44 V. O’Shea,55 F. G. Oakham,31,e H. Oberlack,101 T. Obermann,23 J. Ocariz,81
A. Ochi,68 I. Ochoa,37 J. P. Ochoa-Ricoux,34a S. Oda,71 S. Odaka,67 H. Ogren,62 A. Oh,85 S. H. Oh,47 C. C. Ohm,16
H. Ohman,164 H. Oide,32 H. Okawa,160 Y. Okumura,155 T. Okuyama,67 A. Olariu,28b L. F. Oleiro Seabra,126a
S. A. Olivares Pino,48 D. Oliveira Damazio,27 A. Olszewski,41 J. Olszowska,41 A. Onofre,126a,126e K. Onogi,103
P. U. E. Onyisi,11,w M. J. Oreglia,33 Y. Oren,153 D. Orestano,134a,134b N. Orlando,61b R. S. Orr,158 B. Osculati,52a,52b R. Ospanov,85 G. PRL 117, 111802 (2016) Otero y Garzon,29 H. Otono,71 M. Ouchrif,135d F. Ould-Saada,119 A. Ouraou,136 K. P. Oussoren,107
Q. Ouyang,35a M. Owen,55 R. E. Owen,19 V. E. Ozcan,20a N. Ozturk,8 K. Pachal,142 A. Pacheco Pages,13
136
13
50
16
27
87
119 L. Pacheco Rodriguez,136 C. Padilla Aranda,13 M. Pagáčová,50 S. Pagan Griso,16 F. Paige,27 P. Pais,87 K. Pajchel,119
5 g
,
,
g
,
g
,
g ,
,
j
,
G. Palacino,159b S. Palazzo,39a,39b S. Palestini,32 M. Palka,40b D. Pallin,36 E. St. Panagiotopoulou,10 C. E. Pandini,81
J. G. Panduro Vazquez,78 P. Pani,146a,146b S. Panitkin,27 D. Pantea,28b L. Paolozzi,51 Th. D. Papadopoulou,10
K. Papageorgiou,154 A. Paramonov,6 D. Paredes Hernandez,175 A. J. Parker,73 M. A. Parker,30 K. A. Parker,139
F. Parodi,52a,52b J. A. Parsons,37 U. Parzefall,50 V. R. Pascuzzi,158 E. Pasqualucci,132a S. Passaggio,52a Fr. Pastore,78
G. Pásztor,31,hh S. Pataraia,174 J. R. Pater,85 T. Pauly,32 J. Pearce,168 B. Pearson,113 L. E. Pedersen,38 M. Pedersen,119
S. Pedraza Lopez,166 R. Pedro,126a,126b S. V. Peleganchuk,109,d O. Penc,127 C. Peng,35a H. Peng,35b J. Penwell,62
B. S. Peralva,26b M. M. Perego,136 D. V. Perepelitsa,27 E. Perez Codina,159a L. Perini,92a,92b H. Pernegger,32
S Perrella 104a,104b R Peschke 44 V D Peshekhonov 66 K Peters 44 R F Y Peters 85 B A Petersen 32 T C Petersen 38 g
g
g
g
j
G. Palacino,159b S. Palazzo,39a,39b S. Palestini,32 M. Palka,40b D. Pallin,36 E. St. Panagiotopoulou,10 C. E. Pandini,81
J. G. Panduro Vazquez,78 P. Pani,146a,146b S. Panitkin,27 D. Pantea,28b L. Paolozzi,51 Th. D. Papadopoulou,10
K. Papageorgiou,154 A. Paramonov,6 D. Paredes Hernandez,175 A. J. Parker,73 M. A. Parker,30 K. A. Parker,139
F. Parodi,52a,52b J. A. Parsons,37 U. Parzefall,50 V. R. Pascuzzi,158 E. Pasqualucci,132a S. Passaggio,52a Fr. Pastore,78
G. Pásztor,31,hh S. Pataraia,174 J. R. Pater,85 T. Pauly,32 J. Pearce,168 B. Pearson,113 L. E. Pedersen,38 M. Pedersen,119 B. S. Peralva,26b M. M. Perego,136 D. V. Perepelitsa,27 E. Perez Codina,159a L. Perini,92a,92b H. Pernegger,32
S. Perrella,104a,104b R. Peschke,44 V. D. Peshekhonov,66 K. Peters,44 R. F. Y. Peters,85 B. A. Petersen,32 T. C. Petersen,38 E. Petit,57 A. Petridis,1 C. Petridou,154 P. Petroff,117 E. Petrolo,132a M. Petrov,120 F. Petrucci,134a,134b N. E. Pettersson,87 etit,57 A. Petridis,1 C. Petridou,154 P. Petroff,117 E. Petrolo,132a M. Petrov,120 F. Petrucci,134a,134 A. Peyaud,136 R. Pezoa,34b P. W. Phillips,131 G. Piacquadio,143 E. Pianori,169 A. Picazio,87 E. Piccaro,77 M. Piccinini,22a,22b
M. A. Pickering,120 R. Piegaia,29 J. E. Pilcher,33 A. D. Pilkington,85 A. W. J. Pin,85 M. Pinamonti,163a,163c,ii J. L. Pinfold,3
A. Pingel,38 S. Pires,81 H. PRL 117, 111802 (2016) Manhaes de Andrade Filho,26b
J. Manjarres Ramos,159b A. Mann,100 A. Manousos,32 B. Mansoulie,136 J. D. Mansour,35a R. Mantifel,88 M. Mantoani,56
S. Manzoni,92a,92b L. Mapelli,32 G. Marceca,29 L. March,51 G. Marchiori,81 M. Marcisovsky,127 M. Marjanovic,14
D. E. Marley,90 F. Marroquim,26a S. P. Marsden,85 Z. Marshall,16 S. Marti-Garcia,166 B. Martin,91 T. A. Martin,169
V. J. Martin,48 B. Martin dit Latour,15 M. Martinez,13,s V. I. Martinez Outschoorn,165 S. Martin-Haugh,131 V. S. Martoiu,28b
A. C. Martyniuk,79 M. Marx,138 A. Marzin,32 L. Masetti,84 T. Mashimo,155 R. Mashinistov,96 J. Masik,85
A. L. Maslennikov,109,d I. Massa,22a,22b L. Massa,22a,22b P. Mastrandrea,5 A. Mastroberardino,39a,39b T. Masubuchi,155
P. Mättig,174 J. Mattmann,84 J. Maurer,28b S. J. Maxfield,75 D. A. Maximov,109,d R. Mazini,151 S. M. Mazza,92a,92b
N. C. Mc Fadden,105 G. Mc Goldrick,158 S. P. Mc Kee,90 A. McCarn,90 R. L. McCarthy,148 T. G. McCarthy,101 T. Maier,100 A. Maio,126a,126b,126d S. Majewski,116 Y. Makida,67 N. Makovec,117 B. Malaescu,81 Pa. Malecki,41
V. P. Maleev,123 F. Malek,57 U. Mallik,64 D. Malon,6 C. Malone,143 S. Maltezos,10 S. Malyukov,32 J. Mamuzic,166
G M
i i 49 B M
d lli 32 L M
d lli 92a I M
dić 76 J M
i
126a 126b L M
h
d A d d Filh
26b J. Manjarres Ramos,159b A. Mann,100 A. Manousos,32 B. Mansoulie,136 J. D. Mansour,35a R. Mantifel,88 M. Mantoani,56
S. Manzoni,92a,92b L. Mapelli,32 G. Marceca,29 L. March,51 G. Marchiori,81 M. Marcisovsky,127 M. Marjanovic,14
D. E. Marley,90 F. Marroquim,26a S. P. Marsden,85 Z. Marshall,16 S. Marti-Garcia,166 B. Martin,91 T. A. Martin,169
V. J. Martin,48 B. Martin dit Latour,15 M. Martinez,13,s V. I. Martinez Outschoorn,165 S. Martin-Haugh,131 V. S. Martoiu,28b
A. C. Martyniuk,79 M. Marx,138 A. Marzin,32 L. Masetti,84 T. Mashimo,155 R. Mashinistov,96 J. Masik,85
A L Maslennikov 109,d I Massa 22a,22b L Massa 22a,22b P Mastrandrea 5 A Mastroberardino 39a,39b T Masubuchi 155 111802-10 week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) L. I. McClymont,79 E. F. McDonald,89 J. A. Mcfayden,79 G. Mchedlidze,56 S. J. McMahon M. Medinnis,44 S. Meehan,138 S. Mehlhase,100 A. Mehta,75 K. Meier,59a C. Meineck,100 B. C. Meyer,122 J-P. Meyer,136 J. Meyer,107 H. Meyer Zu Theenhausen,59a F. Miano,149 R. P. Middle L. Mijović,48 G. Mikenberg,171 M. Mikestikova,127 M. Mikuž,76 M. Milesi,89 A. Milic,63 D A. Milov,171 D. A. Milstead,146a,146b A. A. Minaenko,130 Y. Minami,155 I. A. PRL 117, 111802 (2016) Riegel,174 J. Rieger,56 O. Rifki,113 g
g
M. Rijssenbeek,148 A. Rimoldi,121a,121b M. Rimoldi,18 L. Rinaldi,22a B. Ristić,51 E. Ritsch,32 I. Riu,13 F. Rizatdinova,114 E. Rizvi,77 C. Rizzi,13 S. H. Robertson,88,m A. Robichaud-Veronneau,88 D. Robinson,30 J. E. M C. Roda,124a,124b Y. Rodina,86 A. Rodriguez Perez,13 D. Rodriguez Rodriguez,166 S. Roe,32 C. S. Rogan,58 O. Røhne,119
98
22 22b
36
166
138
50
81 a,124b Y. Rodina,86 A. Rodriguez Perez,13 D. Rodriguez Rodriguez,166 S. Roe,32 C. S. Rogan,5
98
22 22b
36
166
138 A. Romaniouk,98 M. Romano,22a,22b S. M. Romano Saez,36 E. Romero Adam,166 N. Rompotis,1 A. Romaniouk,98 M. Romano,22a,22b S. M. Romano Saez,36 E. Romero Adam,166 N. Rompotis,138 M. Ronzani,50 L. Roos,81
E. Ros,166 S. Rosati,132a K. Rosbach,50 P. Rose,137 O. Rosenthal,141 N.-A. Rosien,56 V. Rossetti,146a,146b E. Rossi,104a,104b p
E. Ros,166 S. Rosati,132a K. Rosbach,50 P. Rose,137 O. Rosenthal,141 N.-A. Rosien,56 V. Rossetti,146a,146b E. Rossi,104a,104b L. P. Rossi,52a J. H. N. Rosten,30 R. Rosten,138 M. Rotaru,28b I. Roth,171 J. Rothberg,138 D. Ro A. Rozanov,86 Y. Rozen,152 X. Ruan,145c F. Rubbo,143 M. S. Rudolph,158 F. Rühr,50 A. Ruiz-Martinez,31 Z. Rurikova,50
66
100
138
7
32
123
165 N. A. Rusakovich,66 A. Ruschke,100 H. L. Russell,138 J. P. Rutherfoord,7 N. Ruthmann,32 Y. F. Ryabov,123 M. Rybar,165
G. Rybkin,117 S. Ryu,6 A. Ryzhov,130 G. F. Rzehorz,56 A. F. Saavedra,150 G. Sabato,107 S. Sacerdoti,29 y
y
G. Rybkin,117 S. Ryu,6 A. Ryzhov,130 G. F. Rzehorz,56 A. F. Saavedra,150 G. Sabato,107 S. Sacerdoti,29 H. F-W. Sadrozinski,137 R. Sadykov,66 F. Safai Tehrani,132a P. Saha,108 M. Sahinsoy,59a M. Saimpert,136 T. Saito,155
H. Sakamoto,155 Y. Sakurai,170 G. Salamanna,134a,134b A. Salamon,133a,133b J. E. Salazar Loyola,34b D. Salek,107 H. F W. Sadrozinski,
R. Sadykov,
F. Safai Tehrani,
P. Saha,
M. Sahinsoy,
M. Saimpert,
T. Saito,
H. Sakamoto,155 Y. Sakurai,170 G. Salamanna,134a,134b A. Salamon,133a,133b J. E. Salazar Loyola,34b D. Salek,107 P. H. Sales De Bruin,138 D. Salihagic,101 A. Salnikov,143 J. Salt,166 D. Salvatore,39a,39b F. Sa P. H. Sales De Bruin,138 D. Salihagic,101 A. Salnikov,143 J. Salt,166 D. Salvatore,39a,39b F. Salvatore,149 A. Salvucci,61a
A. Salzburger,32 D. Sammel,50 D. Sampsonidis,154 A. Sanchez,104a,104b J. Sánchez,166 V. Sanchez Martinez,166 A. Salzburger,32 D. Sammel,50 D. Sampsonidis,154 A. Sanchez,104a,104b J. Sánchez,166 V. Sanchez Martinez,166 H. Sandaker,
R. L. Sandbach,
H. G. Sander,
M. Sandhoff,
C. Sandoval,
R. Sandstroem,
D. P. C. Sankey,
M. Sannino,52a,52b A. Sansoni,49 C. Santoni,36 R. Santonico,133a,133b H. Santos,126a I. Santoyo Castillo,149 K. Sapp,125 y
M. Sannino,52a,52b A. Sansoni,49 C. Santoni,36 R. Santonico,133a,133b H. PRL 117, 111802 (2016) Pirumov,44 M. Pitt,171 L. Plazak,144a M.-A. Pleier,27 V. Pleskot,84 E. Plotnikova,66 P. Plucinski,91
D. Pluth,65 R. Poettgen,146a,146b L. Poggioli,117 D. Pohl,23 G. Polesello,121a A. Poley,44 A. Policicchio,39a,39b R. Polifka,158 A. Peyaud,136 R. Pezoa,34b P. W. Phillips,131 G. Piacquadio,143 E. Pianori,169 A. Picazio,87 E. Piccaro,77 M. Piccinini,22a,22b
M. A. Pickering,120 R. Piegaia,29 J. E. Pilcher,33 A. D. Pilkington,85 A. W. J. Pin,85 M. Pinamonti,163a,163c,ii J. L. Pinfold,3
A. Pingel,38 S. Pires,81 H. Pirumov,44 M. Pitt,171 L. Plazak,144a M.-A. Pleier,27 V. Pleskot,84 E. Plotnikova,66 P. Plucinski,91
D. Pluth,65 R. Poettgen,146a,146b L. Poggioli,117 D. Pohl,23 G. Polesello,121a A. Poley,44 A. Policicchio,39a,39b R. Polifka,158
A. Polini,22a C. S. Pollard,55 V. Polychronakos,27 K. Pommès,32 L. Pontecorvo,132a B. G. Pope,91 G. A. Popeneciu,28c
A. Poppleton,32 S. Pospisil,128 K. Potamianos,16 I. N. Potrap,66 C. J. Potter,30 C. T. Potter,116 G. Poulard,32 J. Poveda,32 A. Peyaud,
R. Pezoa,
P. W. Phillips,
G. Piacquadio,
E. Pianori,
A. Picazio,
E. Piccaro,
M. Piccinini,
M. A. Pickering,120 R. Piegaia,29 J. E. Pilcher,33 A. D. Pilkington,85 A. W. J. Pin,85 M. Pinamonti,163a,163c,ii J. L. Pinfold,3 111802-11 week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) S. Prince,88 K. Prokofiev,61c F. Prokoshin,34b S. Protopopescu,27 J. Proudfoot,6 M. Przybyc p p
y y
M. Purohit,27,jj P. Puzo,117 J. Qian,90 G. Qin,55 Y. Qin,85 A. Quadt,56 W. B. Quayle,163a,163b M. Queitsch-Maitland,85
D Quilty 55 S Raddum 119 V Radeka 27 V Radescu 120 S K Radhakrishnan 148 P Radloff 116 P Rados 89 F Ragusa 92a,92b y
G. Rahal,177 J. A. Raine,85 S. Rajagopalan,27 M. Rammensee,32 C. Rangel-Smith,164 M. G. G. Rahal,177 J. A. Raine,85 S. Rajagopalan,27 M. Rammensee,32 C. Rangel-Smith,164 M. G. Ratti,92a,92b F. Rauscher,100
84
55
171
32
119
75
74 74b S. Rave,84 T. Ravenscroft,55 I. Ravinovich,171 M. Raymond,32 A. L. Read,119 N. P. Read D. M. Rebuzzi,121a,121b A. Redelbach,173 G. Redlinger,27 R. Reece,137 K. Reeves,43 L. Rehnisch,17 J. Reichert,122 H. Reisin,29 C. Rembser,32 H. Ren,35a M. Rescigno,132a S. Resconi,92a O. L. Rezanova,109,d P. Reznicek,129 R. Rezvani,95 R. Richter,101
S. Richter,79 E. Richter-Was,40b O. Ricken,23 M. Ridel,81 P. Rieck,17 C. J. Riegel,174 J. Rieger,56 O. Rifki,113 C. Rembser,
H. Ren,
M. Rescigno,
S. Resconi,
O. L. Rezanova,
P. Reznicek,
R. Rezvani,
R. Richter,
S. Richter,79 E. Richter-Was,40b O. Ricken,23 M. Ridel,81 P. Rieck,17 C. J. PRL 117, 111802 (2016) Santos,126a I. Santoyo Castillo,149 K. Sapp,125 A. Sapronov,66 J. G. Saraiva,126a,126d B. Sarrazin,23 O. Sasaki,67 Y. Sasaki,155 K. Sato,160 G. Sauvage,5,a E. Sauvan,5
G. Savage,78 P. Savard,158,e N. Savic,101 C. Sawyer,131 L. Sawyer,80,r J. Saxon,33 C. Sbarra,22a A. Sbrizzi,22a,22b T. Scanlon,79 p
,
,
,
,
,
,
g ,
,
G. Savage,78 P. Savard,158,e N. Savic,101 C. Sawyer,131 L. Sawyer,80,r J. Saxon,33 C. Sbarra,22a A. Sbrizzi,22a,22b T. Scanlon,79
162
150
39a 39b
171
101
100 p
g
G. Savage,78 P. Savard,158,e N. Savic,101 C. Sawyer,131 L. Sawyer,80,r J. Saxon,33 C. Sbarra,22a A. Sbrizzi,22a,22b T. Scanlon,79
D A Scannicchio 162 M Scarcella 150 V Scarfone 39a,39b J Schaarschmidt 171 P Schacht 101 B M Schachtner 100 D. Schaefer,32 L. Schaefer,122 R. Schaefer,44 J. Schaeffer,84 S. Schaepe,23 S. Schaetzel,59b U. Schäfer,84 A. C. Schaffer,117 D. Schaefer,32 L. Schaefer,122 R. Schaefer,44 J. Schaeffer,84 S. Schaepe,23 S. Schaetzel,59b U. S D. Schaefer,32 L. Schaefer,122 R. Schaefer,44 J. Schaeffer,84 S. Schaepe,23 S. Schaetzel,59b U. Schäfer,84 A. C. Schaffer,117
100
148
59a
123
129
162
52a 52b D. Schaile,100 R. D. Schamberger,148 V. Scharf,59a V. A. Schegelsky,123 D. Scheirich,129 M. Sc S. Schier,137 C. Schillo,50 M. Schioppa,39a,39b S. Schlenker,32 K. R. Schmidt-Sommerfeld,101 K. Schmieden,32 C. Schmitt,84
S. Schmitt,44 S. Schmitz,84 B. Schneider,159a U. Schnoor,50 L. Schoeffel,136 A. Schoening,59b B. D. Schoenrock,91 E. Schopf,23 M. Schott,84 J. Schovancova,8 S. Schramm,51 M. Schreyer,173 N. Schuh,84 A. Schulte,84 M. J. Schultens,23
H.-C. Schultz-Coulon,59a H. Schulz,17 M. Schumacher,50 B. A. Schumm,137 Ph. Schune,136 A. Schwartzman,143 T. A. Schwarz,90 H. Schweiger,85 Ph. Schwemling,136 R. Schwienhorst,91 J. Schwindling,136 T. Schwindt,23 G. Sciolla,25
F. Scuri,124a,124b F. Scutti,89 J. Searcy,90 P. Seema,23 S. C. Seidel,105 A. Seiden,137 F. Seifert,128 J. M. Seixas,26a G. Sekhniaidze,104a K. Sekhon,90 S. J. Sekula,42 D. M. Seliverstov,123,a N. Semprini-Cesari,22a,22b C. Serfon,119 L. Serin,117 L. Serkin,163a,163b M. Sessa,134a,134b R. Seuster,168 H. Severini,113 T. Sfiligoj,76 F. Sforza,32 A L. Serkin,163a,163b M. Sessa,134a,134b R. Seuster,168 H. Severini,113 T. Sfiligoj,76 F. Sforza,32 A. Sfyrla,51 E. Shabalina,56
N. W. Shaikh,146a,146b L. Y. Shan,35a R. Shang,165 J. T. Shank,24 M. Shapiro,16 P. B. Shatalov,97 K. Shaw,163a,163b . Se
,
. Sessa,
. Seuste ,
. Seve
,
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goj,
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N. W. Shaikh,146a,146b L. Y. Shan,35a R. Shang,165 J. T. Shank,24 M. Shapiro,16 P. B. Shatalov,97 K. Shaw,163a,163b
85
146 146b
149
9
151 kk
68
162 S. M. Shaw,85 A. PRL 117, 111802 (2016) Shcherbakova,146a,146b C. Y. Shehu,149 P. Sherwood,79 L. Shi,151,kk S. Shimizu,68 C. O. Shimmin,162
M Shi
ji
102 M Shi
k
66,ll A Sh
l
96 D Sh
l h S
di 95 M J Sh
h t 33 S Sh j ii 92a,92b S Sh
th
111 S. M. Shaw,85 A. Shcherbakova,146a,146b C. Y. Shehu,149 P. Sherwood,79 L. Shi,151,kk S. Shimizu,68 C. O. Shimmin,162
M. Shimojima,102 M. Shiyakova,66,ll A. Shmeleva,96 D. Shoaleh Saadi,95 M. J. Shochet,33 S. Shojaii,92a,92b S. Shrestha,111 j
y
j
E. Shulga,98 M. A. Shupe,7 P. Sicho,127 A. M. Sickles,165 P. E. Sidebo,147 O. Sidiropoulou,173 D. Sidorov,114 A. Sidoti,22a,22b E. Shulga,98 M. A. Shupe,7 P. Sicho,127 A. M. Sickles,165 P. E. Sidebo,147 O. Sidiropoulou,173 D F. Siegert,46 Dj. Sijacki,14 J. Silva,126a,126d S. B. Silverstein,146a V. Simak,128 Lj. Simic,14 S F. Siegert,46 Dj. Sijacki,14 J. Silva,126a,126d S. B. Silverstein,146a V. Simak,128 Lj. Simic,14 S. Simion,117 E. Simioni,84
B. Simmons,79 D. Simon,36 M. Simon,84 P. Sinervo,158 N. B. Sinev,116 M. Sioli,22a,22b G. Siragusa,173 S. Yu. Sivoklokov,99
J. Sjölin,146a,146b M. B. Skinner,73 H. P. Skottowe,58 P. Skubic,113 M. Slater,19 T. Slavicek,128 M. Slawinska,107 K. Sliwa,161
R. Slovak,129 V. Smakhtin,171 B. H. Smart,5 L. Smestad,15 J. Smiesko,144a S. Yu. Smirnov,98 Y. Smirnov,98
L. N. Smirnova,99,mm O. Smirnova,82 M. N. K. Smith,37 R. W. Smith,37 M. Smizanska,73 K. Smolek,128 A. A. Snesarev,96
S. Snyder,27 R. Sobie,168,m F. Socher,46 A. Soffer,153 D. A. Soh,151 G. Sokhrannyi,76 C. A. Solans Sanchez,32 M. Solar,128
E. Yu. Soldatov,98 U. Soldevila,166 A. A. Solodkov,130 A. Soloshenko,66 O. V. Solovyanov,130 V. Solovyev,123 P. Sommer,50
H. Son,161 H. Y. Song,35b,nn A. Sood,16 A. Sopczak,128 V. Sopko,128 V. Sorin,13 D. Sosa,59b C. L. Sotiropoulou,124a,124b
R. Soualah,163a,163c A. M. Soukharev,109,d D. South,44 B. C. Sowden,78 S. Spagnolo,74a,74b M. Spalla,124a,124b B. Simmons,79 D. Simon,36 M. Simon,84 P. Sinervo,158 N. B. Sinev,116 M. Sioli,22a,22b G. Siragusa,173 S. Yu. Sivoklokov,99 B. Simmons,79 D. Simon,36 M. Simon,84 P. Sinervo,158 N. B. Sinev,116 M. Sioli,22a,22b G. Si J. Sjölin,146a,146b M. B. Skinner,73 H. P. Skottowe,58 P. Skubic,113 M. Slater,19 T. Slavicek,128 M. Slawinska,107 K. Sliwa,161
R. Slovak,129 V. Smakhtin,171 B. H. Smart,5 L. Smestad,15 J. Smiesko,144a S. Yu. Smirnov,98 Y. Smirnov,98 J. Sjölin,146a,146b M. B. Skinner,73 H. P. Skottowe,58 P. Skubic,113 M. Slater,19 T. Slavicek,128 M. Slawinska,107 K. Sliwa,161
R. Slovak,129 V. Smakhtin,171 B. H. Smart,5 L. Smestad,15 J. Smiesko,144a S. Yu. Smirnov,98 Y. Smirnov,98
L. N. Smirnova,99,mm O. Smirnova,82 M. N. K. Smith,37 R. W. PRL 117, 111802 (2016) Smith,37 M. Smizanska,73 K. Smolek,128 A. A. Snesarev,96 S. Snyder,27 R. Sobie,168,m F. Socher,46 A. Soffer,153 D. A. Soh,151 G. Sokhrannyi,76 C. A. Solans Sanchez,32 M. Solar,128
98
166
130
66
130
123
50 E. Yu. Soldatov,98 U. Soldevila,166 A. A. Solodkov,130 A. Soloshenko,66 O. V. Solovyanov,130 V H. Son,161 H. Y. Song,35b,nn A. Sood,16 A. Sopczak,128 V. Sopko,128 V. Sorin,13 D. Sosa,59b C. L. Sotiropoulou,124a,124b
R. Soualah,163a,163c A. M. Soukharev,109,d D. South,44 B. C. Sowden,78 S. Spagnolo,74a,74b M. Spalla,124a,124b 111802-12 week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) N. Taiblum,153 H. Takai,27 R. Takashima,70 T. Takeshita,140 Y. Takubo,67 M. Talby,86 A. A. Talyshev,109,d K. G. Tan,89 J. Tanaka,155 M. Tanaka,157 R. Tanaka,117 S. Tanaka,67 B. B. Tannenwald,111 S. Tapia Araya,34b S. Tapprogge,84 S. Tarem,152
G. F. Tartarelli,92a P. Tas,129 M. Tasevsky,127 T. Tashiro,69 E. Tassi,39a,39b A. Tavares Delgado,126a,126b Y. Tayalati,135e A. C. Taylor,105 G. N. Taylor,89 P. T. E. Taylor,89 W. Taylor,159b F. A. Teischinger,32 P. Teixeir
142
32
151
118
1 4
6 D. Temple,142 H. Ten Kate,32 P. K. Teng,151 J. J. Teoh,118 F. Tepel,174 S. Terada,67 K. Terashi,155 J. Terron,83 S. Terzo,101
49
158
86
19
8
3 D. Temple,142 H. Ten Kate,32 P. K. Teng,151 J. J. Teoh,118 F. Tepel,174 S. Terada,67 K. Terashi,155 J. Terron,83 S. Terzo,101
M Testa 49 R J Teuscher 158,m T Theveneaux Pelzer 86 J P Thomas 19 J Thomas Wilsker 78 E N Thompson 37 M. Testa,49 R. J. Teuscher,158,m T. Theveneaux-Pelzer,86 J. P. Thomas,19 J. Thomas-Wilsker,78 E. N. Thompson,37
P. D. Thompson,19 A. S. Thompson,55 L. A. Thomsen,175 E. Thomson,122 M. Thomson,30 M. J. Tibbetts,16
R. E. Ticse Torres,86 V. O. Tikhomirov,96,oo Yu. A. Tikhonov,109,d S. Timoshenko,98 P. Tipton,175 S. Tisserant,86 P. D. Thompson,19 A. S. Thompson,55 L. A. Thomsen,175 E. Thomson,122 M. Thomson,30 M. J. Tibbetts,16
86
96
109 d
98
175
86 p
K. Todome,157 T. Todorov,5,a S. Todorova-Nova,129 J. Tojo,71 S. Tokár,144a K. Tokushuku,67 E. Tolley,58 L. Tomlinson,85 j
M. Tomoto,103 L. Tompkins,143,pp K. Toms,105 B. Tong,58 E. Torrence,116 H. Torres,142 E. To F. Touchard,86 D. R. Tovey,139 T. Trefzger,173 A. Tricoli,27 I. M. Trigger,159a S. Trincaz-Duvoid,81 M. F. Tripiana,13
W. Trischuk,158 B. Trocmé,57 A. Trofymov,44 C. Troncon,92a M. Trottier-McDonald,16 M. Trovatelli,168 L. Truong,163a,163c M. Trzebinski,41 A. PRL 117, 111802 (2016) Trzupek,41 J. C-L. Tseng,120 P. V. Tsiareshka,93 G. Tsipolitis,10 N. Tsirintanis,9 S. Tsiskaridze,13
V. Tsiskaridze,50 E. G. Tskhadadze,53a K. M. Tsui,61a I. I. Tsukerman,97 V. Tsulaia,16 S. Tsuno,67 D. Tsybychev,148 Y. Tu,61b y y
A. Tudorache,28b V. Tudorache,28b A. N. Tuna,58 S. A. Tupputi,22a,22b S. Turchikhin,66 D. Turecek,128 D. Turgeman,171
R Turra 92a,92b A J Turvey 42 P M Tuts 37 M Tyndel 131 G Ucchielli 22a,22b I Ueda 155 M Ughetto 146a,146b F Ukegawa 160 y y
A. Tudorache,28b V. Tudorache,28b A. N. Tuna,58 S. A. Tupputi,22a,22b S. Turchikhin,66 D. Turecek,128 D. Turgeman,171 R. Turra,92a,92b A. J. Turvey,42 P. M. Tuts,37 M. Tyndel,131 G. Ucchielli,22a,22b I. Ueda,155 M. Ugh G. Unal,32 A. Undrus,27 G. Unel,162 F. C. Ungaro,89 Y. Unno,67 C. Unverdorben,100 J. Urban,144b P. Urquijo,89 P. Urrejola,84
G. Usai,8 A. Usanova,63 L. Vacavant,86 V. Vacek,128 B. Vachon,88 C. Valderanis,100 E. Valdes Santurio,146a,146b N. Valencic,107 S. Valentinetti,22a,22b A. Valero,166 L. Valery,13 S. Valkar,129 J. A. Valls Ferrer,166 W. Van Den Wollenberg,107
5 N. Valencic,107 S. Valentinetti,22a,22b A. Valero,166 L. Valery,13 S. Valkar,129 J. A. Valls Ferrer,166 W. Van Den Wollenberg,107
P C Van Der Deijl 107 H van der Graaf 107 N van Eldik 152 P van Gemmeren 6 J Van Nieuwkoop 142 I van Vulpen 107 P. C. Van Der Deijl,107 H. van der Graaf,107 N. van Eldik,152 P. van Gemmeren,6 J. Van Nieuw M. C. van Woerden,32 M. Vanadia,132a,132b W. Vandelli,32 R. Vanguri,122 A. Vaniachine,130 P. Vankov,107 G. Vardanyan,176
132
7
42
81
8
150
175
36 R. Vari,132a E. W. Varnes,7 T. Varol,42 D. Varouchas,81 A. Vartapetian,8 K. E. Varvell,150 J. G. Vasquez,175 F. Vazeille,36
T. Vazquez Schroeder,88 J. Veatch,56 V. Veeraraghavan,7 L. M. Veloce,158 F. Veloso,126a,126c S. Veneziano,132a
74 74b
168
158
25
121
132 132b
107 R. Vari,132a E. W. Varnes,7 T. Varol,42 D. Varouchas,81 A. Vartapetian,8 K. E. Varvell,150 J. G. Vasquez,175 F. Vazeille,36
T. Vazquez Schroeder,88 J. Veatch,56 V. Veeraraghavan,7 L. M. Veloce,158 F. Veloso,126a,126c S. Veneziano,132a
A. Ventura,74a,74b M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke,107
J C V
l
107 A V
46 rr M C V
li 142 e O Vi
l
82 I Vi h
165 a T Vi k
139 O E Vi k
B
i
139 q
g
A. Ventura,74a,74b M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke,107
J. C. Vermeulen,107 A. Vest,46,rr M. C. Vetterli,142,e O. PRL 117, 111802 (2016) Webb,84 M. S. Weber,18 S. W. Weber,173 J. S. Webster,6 A. R. Weidberg,120 B. Weinert,62 J. Weingarten,56
C. Weiser,50 H. Weits,107 P. S. Wells,32 T. Wenaus,27 T. Wengler,32 S. Wenig,32 N. Wermes,23 M. Werner,50 M. D. Werner,65
P. Werner,32 M. Wessels,59a J. Wetter,161 K. Whalen,116 N. L. Whallon,138 A. M. Wharton,73 A. White,8 M. J. White,1
R. White,34b D. Whiteson,162 F. J. Wickens,131 W. Wiedenmann,172 M. Wielers,131 P. Wienemann,23 C. Wiglesworth,38
L. A. M. Wiik-Fuchs,23 A. Wildauer,101 F. Wilk,85 H. G. Wilkens,32 H. H. Williams,122 S. Williams,107 C. Willis,91
S. Willocq,87 J. A. Wilson,19 I. Wingerter-Seez,5 F. Winklmeier,116 O. J. Winston,149 B. T. Winter,23 M. Wittgen,143 R. Wang,6 S. M. Wang,151 T. Wang,23 T. Wang,37 W. Wang,35b X. Wang,175 C. Wanotayaroj,116 A. Warburton,88 C. P. Ward,30
D. R. Wardrope,79 A. Washbrook,48 P. M. Watkins,19 A. T. Watson,19 M. F. Watson,19 G. Watts,138 S. Watts,85 D. R. Wardrope,79 A. Washbrook,48 P. M. Watkins,19 A. T. Watson,19 M. F. Watson,19 G. Watts,138 S. Watts,85
B. M. Waugh,79 S. Webb,84 M. S. Weber,18 S. W. Weber,173 J. S. Webster,6 A. R. Weidberg,120 B. Weinert,62 J. Weingarten,56
50
107
32
27
32
32
23
50
65 p ,
,
,
,
,
,
,
B. M. Waugh,79 S. Webb,84 M. S. Weber,18 S. W. Weber,173 J. S. Webster,6 A. R. Weidberg,120 B. Weinert,62 J. Weingarten,56 111802-13 PRL 117, 111802 (2016) Viazlo,82 I. Vichou,165,a T. Vickey,139 O. E. Vickey Boeriu,139
120
16
120
22a 22b
92a 92b
49
31 g
M. Venturi,168 N. Venturi,158 A. Venturini,25 V. Vercesi,121a M. Verducci,132a,132b W. Verkerke, A. Ventura,
M. Venturi,
N. Venturi,
A. Venturini,
V. Vercesi,
M. Verducci,
W. Verkerke,
J. C. Vermeulen,107 A. Vest,46,rr M. C. Vetterli,142,e O. Viazlo,82 I. Vichou,165,a T. Vickey,139 O. E. Vickey Boeriu,139
120
16
120
22 22b
92 92b
49
31 J. C. Vermeulen,107 A. Vest,46,rr M. C. Vetterli,142,e O. Viazlo,82 I. Vichou,165,a T. Vickey,139 O G. H. A. Viehhauser,120 S. Viel,16 L. Vigani,120 M. Villa,22a,22b M. Villaplana Perez,92a,92b E. V g
p
V. B. Vinogradov,66 C. Vittori,22a,22b I. Vivarelli,149 S. Vlachos,10 M. Vlasak,128 M. Vogel,174 P. Vokac,128 G. Volpi,124a,124b M. Volpi,89 H. von der Schmitt,101 E. von Toerne,23 V. Vorobel,129 K. Vorobev,98 M. Vos,166 R. Voss,32 J. H. Vossebeld,75
14
14
127
107
32
33
128 M. Volpi,89 H. von der Schmitt,101 E. von Toerne,23 V. Vorobel,129 K. Vorobev,98 M. Vos,166 R. Voss,32 J. H. Vossebeld,75
N. Vranjes,14 M. Vranjes Milosavljevic,14 V. Vrba,127 M. Vreeswijk,107 R. Vuillermet,32 I. Vukotic,33 Z. Vykydal,128 mitt,101 E. von Toerne,23 V. Vorobel,129 K. Vorobev,98 M. Vos,166 R. Voss,32 J. H. Vossebeld,75 j
,
j
j
,
,
j ,
,
,
y y
,
P. Wagner,23 W. Wagner,174 H. Wahlberg,72 S. Wahrmund,46 J. Wakabayashi,103 J. Walder,73 R. Walker,100 W. Walkowiak,141
V. Wallangen,146a,146b C. Wang,35c C. Wang,35d,86 F. Wang,172 H. Wang,16 H. Wang,42 J. Wang,44 J. Wang,150 K. Wang,88
R. Wang,6 S. M. Wang,151 T. Wang,23 T. Wang,37 W. Wang,35b X. Wang,175 C. Wanotayaroj,116 A. Warburton,88 C. P. Ward,30
D R Wardrope 79 A Washbrook 48 P M Watkins 19 A T Watson 19 M F Watson 19 G Watts 138 S Watts 85 j
,
j
j
,
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j ,
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,
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,
P. Wagner,23 W. Wagner,174 H. Wahlberg,72 S. Wahrmund,46 J. Wakabayashi,103 J. Walder,73 R. Walker,100 W. Walkowiak,141
V W ll
146a,146b C W
35c C W
35d,86 F W
172 H W
16 H W
42 J W
44 J W
150 K W
88 g
,
g,
g,
g,
g,
g,
g,
g,
g,
R. Wang,6 S. M. Wang,151 T. Wang,23 T. Wang,37 W. Wang,35b X. Wang,175 C. Wanotayaroj,116 A. Warburton,88 C. P. Ward,30
D. R. Wardrope,79 A. Washbrook,48 P. M. Watkins,19 A. T. Watson,19 M. F. Watson,19 G. Watts,138 S. Watts,85
B. M. Waugh,79 S. 111802-13 week ending
9 SEPTEMBER 2016 week ending
9 SEPTEMBER 2016 P H Y S I C A L
R E V I E W
L E T T E R S PRL 117, 111802 (2016) J. Wittkowski,100 T. M. H. Wolf,107 M. W. Wolter,41 H. Wolters,126a,126c S. D. Worm,131 B. K. Wosiek,41 J. Wotschack,32
M. J. Woudstra,85 K. W. Wozniak,41 M. Wu,57 M. Wu,33 S. L. Wu,172 X. Wu,51 Y. Wu,90 T. R. Wyatt,85 B. M. Wynne,48
S. Xella,38 D. Xu,35a L. Xu,27 B. Yabsley,150 S. Yacoob,145a D. Yamaguchi,157 Y. Yamaguchi,118 A. Yamamoto,67
S. Yamamoto,155 T. Yamanaka,155 K. Yamauchi,103 Y. Yamazaki,68 Z. Yan,24 H. Yang,35e H. Yang,172 Y. Yang,151 Z. Yang,15
W-M. Yao,16 Y. C. Yap,81 Y. Yasu,67 E. Yatsenko,5 K. H. Yau Wong,23 J. Ye,42 S. Ye,27 I. Yeletskikh,66 A. L. Yen,58
E. Yildirim,84 K. Yorita,170 R. Yoshida,6 K. Yoshihara,122 C. Young,143 C. J. S. Young,32 S. Youssef,24 D. R. Yu,16 J. Yu,8
J. M. Yu,90 J. Yu,65 L. Yuan,68 S. P. Y. Yuen,23 I. Yusuff,30,ss B. Zabinski,41 R. Zaidan,35d A. M. Zaitsev,130,ff N. Zakharchuk,44
J. Zalieckas,15 A. Zaman,148 S. Zambito,58 L. Zanello,132a,132b D. Zanzi,89 C. Zeitnitz,174 M. Zeman,128 A. Zemla,40a
J. C. Zeng,165 Q. Zeng,143 K. Zengel,25 O. Zenin,130 T. Ženiš,144a D. Zerwas,117 D. Zhang,90 F. Zhang,172 G. Zhang,35b,nn
H. Zhang,35c J. Zhang,6 L. Zhang,50 R. Zhang,23 R. Zhang,35b,tt X. Zhang,35d Z. Zhang,117 X. Zhao,42 Y. Zhao,35d Z. Zhao,35b
A. Zhemchugov,66 J. Zhong,120 B. Zhou,90 C. Zhou,47 L. Zhou,37 L. Zhou,42 M. Zhou,148 N. Zhou,35f C. G. Zhu,35d
H. Zhu,35a J. Zhu,90 Y. Zhu,35b X. Zhuang,35a K. Zhukov,96 A. Zibell,173 D. Zieminska,62 N. I. Zimine,66 C. Zimmermann,84
S. Zimmermann,50 Z. Zinonos,56 M. Zinser,84 M. Ziolkowski,141 L. Živković,14 G. Zobernig,172 A. Zoccoli,22a,22b
M zur Nedden 17 and L Zwalinski32 M. zur Nedden,17 and L. Zwalinski32 J. Wittkowski,100 T. M. H. Wolf,107 M. W. Wolter,41 H. Wolters,126a,126c S. D. Worm,131 B. K. Wosiek,41 J. Wotschack,32
M. J. Woudstra,85 K. W. Wozniak,41 M. Wu,57 M. Wu,33 S. L. Wu,172 X. Wu,51 Y. Wu,90 T. R. Wyatt,85 B. M. Wynne,48
S. Xella,38 D. Xu,35a L. Xu,27 B. Yabsley,150 S. Yacoob,145a D. Yamaguchi,157 Y. Yamaguchi,118 A. Yamamoto,67
S. Yamamoto,155 T. Yamanaka,155 K. Yamauchi,103 Y. Yamazaki,68 Z. Yan,24 H. Yang,35e H. Yang,172 Y. Yang,151 Z. Yang,15
W-M. Yao,16 Y. C. Yap,81 Y. Yasu,67 E. Yatsenko,5 K. H. Yau Wong,23 J. Ye,42 S. Ye,27 I. Yeletskikh,66 A. L. Yen,58
E. Yildirim,84 K. Yorita,170 R. Yoshida,6 K. Yoshihara,122 C. Young,143 C. J. S. Young,32 S. Youssef,24 D. R. Yu,16 J. Yu,8
J. M. Yu,90 J. Yu,65 L. Yuan,68 S. P. Y. Yuen,23 I. Yusuff,30,ss B. Zabinski,41 R. Zaidan,35d A. M. Zaitsev,130,ff N. Zakharchuk,44
J. Zalieckas,15 A. Zaman,148 S. Zambito,58 L. Zanello,132a,132b D. Zanzi,89 C. Zeitnitz,174 M. Zeman,128 A. Zemla,40a
J. C. Zeng,165 Q. Zeng,143 K. Zengel,25 O. Zenin,130 T. Ženiš,144a D. Zerwas,117 D. Zhang,90 F. Zhang,172 G. Zhang,35b,nn
H. Zhang,35c J. Zhang,6 L. Zhang,50 R. Zhang,23 R. Zhang,35b,tt X. Zhang,35d Z. Zhang,117 X. Zhao,42 Y. Zhao,35d Z. Zhao,35b
A. Zhemchugov,66 J. Zhong,120 B. Zhou,90 C. Zhou,47 L. Zhou,37 L. Zhou,42 M. Zhou,148 N. Zhou,35f C. G. Zhu,35d
H. Zhu,35a J. Zhu,90 Y. Zhu,35b X. Zhuang,35a K. Zhukov,96 A. Zibell,173 D. Zieminska,62 N. I. Zimine,66 C. Zimmermann,84
S. Zimmermann,50 Z. Zinonos,56 M. Zinser,84 M. Ziolkowski,141 L. Živković,14 G. Zobernig,172 A. Zoccoli,22a,22b
M zur Nedden 17 and L Zwalinski32 (ATLAS Collaboration) 1Department of Physics, University of Adelaide, Adelaide, Australia
2Physics Department, SUNY Albany, Albany, New York, USA
3Department of Physics, University of Alberta, Edmonton, Alberta, Canada
4aDepartment of Physics, Ankara University, Ankara, Turkey
4bIstanbul Aydin University, Istanbul, Turkey
4cDivision of Physics, TOBB University of Economics and Technology, Ankara, Turkey
5LAPP, CNRS/IN2P3 and Université Savoie Mont Blanc, Annecy-le-Vieux, France
6High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA
7Department of Physics, University of Arizona, Tucson, Arizona, USA
8Department of Physics, The University of Texas at Arlington, Arlington, Texas, USA
9Physics Department, University of Athens, Athens, Greece
10Physics Department, National Technical University of Athens, Zografou, Greece
11Department of Physics, The University of Texas at Austin, Austin, Texas, USA
12Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan
13Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain
14Institute of Physics, University of Belgrade, Belgrade, Serbia
15Department for Physics and Technology, University of Bergen, Bergen, Norway
16Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, California, USA
17Department of Physics, Humboldt University, Berlin, Germany
18Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland
19School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom
20aDepartment of Physics, Bogazici University, Istanbul, Turkey
20bDepartment of Physics Engineering, Gaziantep University, Gaziantep, Turkey
20cIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey
20dBahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey
21Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia
22aINFN Sezione di Bologna, Italy
22bDipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy
23Physikalisches Institut, University of Bonn, Bonn, Germany
24Department of Physics, Boston University, Boston, Massachusetts, USA
25Department of Physics, Brandeis University, Waltham, Massachusetts, USA
26aUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil
26bElectrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora, Brazil
26cFederal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei, Brazil
26dInstituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil
27Physics Department, Brookhaven National Laboratory, Upton, New York, USA
28aTransilvania University of Brasov, Brasov, Romania, Romania
28bNational Institute of Physics and Nuclear Engineering, Bucharest, Romania 1Department of Physics, University of Adelaide, Adelaide, Australia
2 1Department of Physics, University of Adelaide, Adelaide, Australia
2Physics Department, SUNY Albany, Albany, New York, USA
3Department of Physics, University of Alberta, Edmonton, Alberta, Canada
4aDepartment of Physics, Ankara University, Ankara, Turkey
4bIstanbul Aydin University, Istanbul, Turkey
4cDivision of Physics, TOBB University of Economics and Technology, Ankara, Turkey
5LAPP, CNRS/IN2P3 and Université Savoie Mont Blanc, Annecy-le-Vieux, France
6High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA
7Department of Physics, University of Arizona, Tucson, Arizona, USA
8Department of Physics, The University of Texas at Arlington, Arlington, Texas, USA
9Physics Department, University of Athens, Athens, Greece
10Physics Department, National Technical University of Athens, Zografou, Greece
11Department of Physics, The University of Texas at Austin, Austin, Texas, USA
12Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan
13Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain
14Institute of Physics, University of Belgrade, Belgrade, Serbia
15Department for Physics and Technology, University of Bergen, Bergen, Norway
16Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, California, USA
17Department of Physics, Humboldt University, Berlin, Germany
18Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland
19School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom
20aDepartment of Physics, Bogazici University, Istanbul, Turkey
20bDepartment of Physics Engineering, Gaziantep University, Gaziantep, Turkey
20cIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey
20dBahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey
21Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia
22aINFN Sezione di Bologna, Italy
22bDipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy
23Physikalisches Institut, University of Bonn, Bonn, Germany
24Department of Physics, Boston University, Boston, Massachusetts, USA
25Department of Physics, Brandeis University, Waltham, Massachusetts, USA
26aUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil
26bElectrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora, Brazil
26cFederal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei, Brazil
26dInstituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil
27Physics Department, Brookhaven National Laboratory, Upton, New York, USA
28aTransilvania University of Brasov, Brasov, Romania, Romania
28bNational Institute of Physics and Nuclear Engineering, Bucharest, Romania 2Physics Department, SUNY Albany, Albany, New York, USA 3Department of Physics, University of Alberta, Edmonton, Alberta, Canada
4 4aDepartment of Physics, Ankara University, Ankara, Turkey 4bIstanbul Aydin University, Istanbul, Turkey al Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 2aINFN Sezione di Bologna, Italy 28bNational Institute of Physics and Nuclear Engineering, Bucharest, Romania 111802-14 111802-14 52aINFN Sezione di Genova, Italy 52bDipartimento di Fisica, Università di Genova, Genova, Italy shvili Institute of Physics, Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia
b 53bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia
54 54II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 111802-15 44DESY, Hamburg and Zeuthen, Germany 45Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany
46 45Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany
46 47Department of Physics, Duke University, Durham, North Carolina, USA 48SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United
49 49INFN Laboratori Nazionali di Frascati, Frascati, Italy
50 50Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany
51 51Section de Physique, Université de Genève, Geneva, Switzerland
52 111802-14 Andronikashvili Institute of Physics, Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia
53bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia
54II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany
55SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom
56II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany
57Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France
58Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA
59aKirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany
59bPhysikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany
59cZITI Institut für technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany
60Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan
61aDepartment of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
61bDepartment of Physics, The University of Hong Kong, Hong Kong, China
epartment of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, C
62Department of Physics, Indiana University, Bloomington, Indiana, USA
63Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria
64University of Iowa, Iowa City, Iowa, USA
65Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA
66Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia
67KEK, High Energy Accelerator Research Organization, Tsukuba, Japan
68Graduate School of Science, Kobe University, Kobe, Japan
L 117, 111802 (2016)
9 SEPTEMBER 28cNational Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca, Romania
28d 36Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 37Nevis Laboratory, Columbia University, Irvington, New York, USA
38 38Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 9aINFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy
39b 39bDipartimento di Fisica, Università della Calabria, Rende, Italy 0aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland
40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 40aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland
40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland
41 AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland
40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland
41 41Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland
42 42Physics Department, Southern Methodist University, Dallas, Texas, USA
3 42Physics Department, Southern Methodist University, Dallas, Texas, USA
3 43Physics Department, University of Texas at Dallas, Richardson, Texas, USA
44 43Physics Department, University of Texas at Dallas, Richardson, Texas, USA
44 111802-14 week ending
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Physics Department, Cluj Napoca, Romania
28dUniversity Politehnica Bucharest, Bucharest, Romania
28eWest University in Timisoara, Timisoara, Romania
29Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina
30Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom
31Department of Physics, Carleton University, Ottawa, Ontario, Canada
32CERN, Geneva, Switzerland
33Enrico Fermi Institute, University of Chicago, Chicago, Illinois, USA
34aDepartamento de Física, Pontificia Universidad Católica de Chile, Santiago, Chile
34bDepartamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile
35aInstitute of High Energy Physics, Chinese Academy of Sciences, Beijing, China
35bDepartment of Modern Physics, University of Science and Technology of China, Anhui, China
35cDepartment of Physics, Nanjing University, Jiangsu, China
35dSchool of Physics, Shandong University, Shandong, China
35eDepartment of Physics and Astronomy, Shanghai Key Laboratory for Particle Physics and Cosmology,
Shanghai Jiao Tong University, Shanghai; (also affiliated with PKU-CHEP), China
35fPhysics Department, Tsinghua University, Beijing 100084, China
36Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3,
Clermont-Ferrand, France
37Nevis Laboratory, Columbia University, Irvington, New York, USA
38Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark
39aINFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy
39bDipartimento di Fisica, Università della Calabria, Rende, Italy
40aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland
40bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland
41Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland
42Physics Department, Southern Methodist University, Dallas, Texas, USA
43Physics Department, University of Texas at Dallas, Richardson, Texas, USA
44DESY, Hamburg and Zeuthen, Germany
45Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany
46Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany
47Department of Physics, Duke University, Durham, North Carolina, USA
48SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom
49INFN Laboratori Nazionali di Frascati, Frascati, Italy
50Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany
51Section de Physique, Université de Genève, Geneva, Switzerland
52aINFN Sezione di Genova, Italy
52bDipartimento di Fisica, Università di Genova, Genova, Italy
53aE. P H Y S I C A L
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L E T T E R S PRL 117, 111802 (2016) 69Faculty of Science, Kyoto University, Kyoto, Japan
70Kyoto University of Education, Kyoto, Japan
71Department of Physics, Kyushu University, Fukuoka, Japan
72Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina
73Physics Department, Lancaster University, Lancaster, United Kingdom
74aINFN Sezione di Lecce, Italy
74bDipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy
75Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom
76Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljubljana, Slovenia
77School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom
78Department of Physics, Royal Holloway University of London, Surrey, United Kingdom
79Department of Physics and Astronomy, University College London, London, United Kingdom
80Louisiana Tech University, Ruston, Louisiana, USA
81Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, Fr
82Fysiska institutionen, Lunds universitet, Lund, Sweden
83Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain
84Institut für Physik, Universität Mainz, Mainz, Germany
85School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom
86CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France
87Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA
88Department of Physics, McGill University, Montreal, Québec, Canada
89School of Physics, University of Melbourne, Victoria, Australia
90Department of Physics, The University of Michigan, Ann Arbor, Michigan, USA
91Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA
92aINFN Sezione di Milano, Italy
92bDipartimento di Fisica, Università di Milano, Milano, Italy
93B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus
94National Scientific and Educational Centre for Particle and High Energy Physics, Minsk, Republic of Belarus
95Group of Particle Physics, University of Montreal, Montreal, Québec, Canada
96P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia
97Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia
98National Research Nuclear University MEPhI, Moscow, Russia
99D.V. Skobeltsyn Institute of Nuclear Physics, M.V. P H Y S I C A L
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L E T T E R S Lomonosov Moscow State University, Moscow, Russia
100Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany
101Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany
102Nagasaki Institute of Applied Science, Nagasaki, Japan
103Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan
104aINFN Sezione di Napoli, Italy
104bDipartimento di Fisica, Università di Napoli, Napoli, Italy
105Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA
106Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherland
107Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands
108Department of Physics, Northern Illinois University, DeKalb, Illinois, USA
109Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia
110Department of Physics, New York University, New York, New York, USA
111Ohio State University, Columbus, Ohio, USA
112Faculty of Science, Okayama University, Okayama, Japan
113Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA
114Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA
115Palacký University, RCPTM, Olomouc, Czech Republic
116Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA
117LAL, Université Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France
118Graduate School of Science, Osaka University, Osaka, Japan
119Department of Physics, University of Oslo, Oslo, Norway
120Department of Physics, Oxford University, Oxford, United Kingdom
121aINFN Sezione di Pavia, Italy
121bDipartimento di Fisica, Università di Pavia, Pavia, Italy
122Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA
123National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Ru
124aINFN Sezione di Pisa, Italy
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9 SEPTEMBER 70Kyoto University of Education, Kyoto, Japan 81Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France
82 de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain
84 cs and Astronomy, University of Manchester, Manchester, United Kingdom 88Department of Physics, McGill University, Montreal, Québec, Canada
89 89School of Physics, University of Melbourne, Victoria, Australia 90Department of Physics, The University of Michigan, Ann Arbor, Michigan, USA 90Department of Physics, The University of Michigan, Ann Arbor, Michigan, USA rtment of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA
92 Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA
92 111802-15 week ending
9 SEPTEMBER 2016 92aINFN Sezione di Milano, Italy ento di Fisica, Università di Milano, Milano, Italy 92bDipartimento di Fisica, Università di Milano, Milano, Italy 100Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany
101 101Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany
102 102Nagasaki Institute of Applied Science, Nagasaki, Japan 3Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan
104a Casablanca, Morocco 35bCentre National de l’Energie des Sciences Techniques Nucleaires, Rabat, Morocco
5 135cFaculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech, Morocc
135d 5dFaculté des Sciences, Université Mohamed Premier and LPTPM, Oujda, Morocco
135 135eFaculté des sciences, Université Mohammed V, Rabat, Morocco sciences, Université Mohammed V, Rabat, Morocco erches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et 136DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commis aux Energies Alternatives), Gif-sur-Yvette, France aux Energies Alternatives), Gif-sur-Yvette, France aux Energies Alternatives), Gif-sur-Yvette, France g
f
ute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA g
f
Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA
138 137Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, Cal
138 ticle Physics, University of California Santa Cruz, 138Department of Physics, University of Washington, Seattle, Washington, USA 139Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom
140 139Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom rtment of Physics, Shinshu University, Nagano, Jap 141Fachbereich Physik, Universität Siegen, Siegen, Germany 141Fachbereich Physik, Universität Siegen, Siegen, Germany of Physics, Simon Fraser University, Burnaby, British Columbia, Canada 143SLAC National Accelerator Laboratory, Stanford, California, USA 144aFaculty of Mathematics, Physics & Informatics, Comenius University, Bratislava, Slovak Republic
144b p
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145aDepartment of Physics, University of Cape Town, Cape Town, South Africa
145b 145bDepartment of Physics, University of Johannesburg, Johannesburg, South Africa
145 145cSchool of Physics, University of the Witwatersrand, Johannesburg, South Africa
146 46aDepartment of Physics, Stockholm University, Sweden
146b 46bThe Oskar Klein Centre, Stockholm, Sweden 147Physics Department, Royal Institute of Technology, Stockholm, Sweden 149Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom
150 150School of Physics, University of Sydney, Sydney, Australia
151 151Institute of Physics, Academia Sinica, Taipei, Taiwan 152Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 154Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece for Elementary Particle Physics and Department of Physics, The University of Tokyo, T
uate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan
157 157Department of Physics, Tokyo Institute of Technology, Tokyo, Japan
8 158Department of Physics, University of Toronto, Toronto, Ontario, Canada
159 159aTRIUMF, Vancouver, British Columbia, Canada 59bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada
and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering 159bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada
160Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, p
f
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160Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, University of Tsukuba, Tsukuba, Japan Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA Department of Physics and Astronomy, University of California Irvine, Irvine, California, USA 111802-17 4aINFN Sezione di Napoli, Italy 104bDipartimento di Fisica, Università di Napoli, Napoli, Italy 5Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA
r Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Ne l Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 108Department of Physics, Northern Illinois University, DeKalb, Illinois, USA
109 e of Nuclear Physics, SB RAS, Novosibirsk, Russia 110Department of Physics, New York University, New York, New York, USA
111 111Ohio State University, Columbus, Ohio, USA 112Faculty of Science, Okayama University, Okayama, Japan 113Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA
114D
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USA dge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA
4 114Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA
115 115Palacký University, RCPTM, Olomouc, Czech Republic
116 115Palacký University, RCPTM, Olomouc, Czech Republic
116 cký University, RCPTM, Olomouc, Czech Republic 116Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA
117 117LAL, Université Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France
118 118Graduate School of Science, Osaka University, Osaka, Japan
119 119Department of Physics, University of Oslo, Oslo, Norway 120Department of Physics, Oxford University, Oxford, United Kingdom
121 122Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA
123National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Russia
124aINFN Sezione di Pisa, Italy 111802-16 week ending
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132bDipartimento di Fisica, Sapienza Università di Roma, Roma, Italy
133 133aINFN Sezione di Roma Tor Vergata, Italy 133bDipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy
134 bDipartimento di Matematica e Fisica, Università R p
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135aFaculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Uni 135aFaculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, P H Y S I C A L
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L E T T E R S wAlso at Department of Physics, The University of Texas at Austin, Austin TX, US
x te of Theoretical Physics, Ilia State University, Tbil xAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. retical Physics, Ilia State University, Tbilisi, Georgi yAlso at CERN, Geneva, Switzerland. yAlso at CERN, Geneva, Switzerland. zAlso at Georgian Technical University (GTU),Tbilisi, Georgia. zAlso at Georgian Technical University (GTU),Tbilisi, Georgia. zAlso at Georgian Technical University aaAlso at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. bb aaAlso at Ochadai Academic Production, Ochanomizu University, To
bb bbAlso at Manhattan College, New York NY, USA. bbAlso at Manhattan College, New York NY, USA. cAlso at Hellenic Open University, Patras, Greece. d p
y
ddAlso at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. eeAlso at School of Physics, Shandong University, Shandong, China. ff eeAlso at School of Physics, Shandong University, Shandong, China. ff ffAlso at Moscow Institute of Physics and Technology State University, Dolgoprudny, Ru ggAlso at Section de Physique, Université de Genève, Geneva, Switzerland. hh o at Section de Physique, Université de Genève, Ge hhAlso at Eotvos Lorand University, Budapest, Hungary. jjAlso at Department of Physics and Astronomy, University of South Carolina, Columbia SC, USA. kk jjAlso at Department of Physics and Astronomy, University of South Carolina, Columbia SC, USA. kk Department of Physics and Astronomy, University o kkAlso at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China. ll y
g
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llAlso at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Sofia, Bulgaria. mmAlso at Faculty of Physics M VLomonosov Moscow State University Moscow Russia llAlso at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Aca mmAlso at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia. 111802-18 P H Y S I C A L
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L E T T E R S fAlso at Department of Physics & Astronomy, University of Louisville, Louisville, KY, USA. hysics, California State University, Fresno CA, USA hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i iAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain. j iAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barce
j jAlso at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Universidade do Porto, Portug
k jAlso at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Unive
k jAlso at Departamento de Fisica e Astronomia, Fac
k kAlso at Tomsk State University, Tomsk, Russia. kAlso at Tomsk State University, Tomsk, Russia. lAlso at Universita di Napoli Parthenope, Napoli, I mAlso at Institute of Particle Physics (IPP), Canada mAlso at Institute of Particle Physics (IPP), Canada. nAlso at National Institute of Physics and Nuclear Engineering, Bucharest, Romania. nAlso at National Institute of Physics and Nuclear Engineering, Bucharest, Romania oAlso at Department of Physics, St. Petersburg State Polytechnical University, St. Pe oAlso at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russi pAlso at Department of Physics, The University of Michigan, Ann Arbor MI, USA. pAlso at Department of Physics, The University of Michigan, Ann Arbor MI, USA. qAlso at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Afr Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa. rAlso at Louisiana Tech University, Ruston LA, USA. rAlso at Louisiana Tech University, Ruston LA, USA. rAlso at Louisiana Tech University, Ruston LA, USA. Also at Louisiana Tech University, Ruston LA, USA y
sAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. t tAlso at Graduate School of Science, Osaka University, Osaka, Japan. tAlso at Graduate School of Science, Osaka University, Osaka, Japan. uAlso at Department of Physics, National Tsing Hua University, Taiwan. uAlso at Department of Physics, National Tsing Hua University, Taiwa vAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen,
Netherlands. vAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen,
Netherlands. vAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud Universi
Netherlands. 111802-17 week ending
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L E T T E R S PRL 117, 111802 (2016) 163aINFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy 163aINFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy
163bICTP, Trieste, Italy
163cDipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy
164Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden
165Department of Physics, University of Illinois, Urbana, Illinois, USA
166Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica,
Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM),
University of Valencia and CSIC, Valencia, Spain
167Department of Physics, University of British Columbia, Vancouver, British Columbia, Canada
168Department of Physics and Astronomy, University of Victoria, Victoria, British Columbia, Canada
169Department of Physics, University of Warwick, Coventry, United Kingdom
170Waseda University, Tokyo, Japan
171Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel
172Department of Physics, University of Wisconsin, Madison, Wisconsin, USA
173Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany
174Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany
175Department of Physics, Yale University, New Haven, Connecticut, USA
176 165Department of Physics, University of Illinois, Urbana, Illinois, USA
66 166Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Department of Physics, University of Warwick, Coventry, United Kingdom
170 71Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel
172 172Department of Physics, University of Wisconsin, Madison, Wisconsin, USA
173 173Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany thematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal
175 f
f
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175Department of Physics, Yale University, New Haven, Connecticut, USA
176 175Department of Physics, Yale University, New Haven, Connecticut, USA
176 176Yerevan Physics Institute, Yerevan, Armenia 76Yerevan Physics Institute, Yerevan, Armenia 177Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3 177Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France aDeceased. b bAlso at Department of Physics, King’s College London, London, United Kingdom. bAlso at Department of Physics, King’s College London, London, United Kingdom. cAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. d dAlso at Novosibirsk State University, Novosibirsk, Russia. dAlso at Novosibirsk State University, Novosibirsk, Russia. eAlso at TRIUMF, Vancouver BC, Canada. f eAlso at TRIUMF, Vancouver BC, Canada. f fAlso at Department of Physics & Astronomy, University of Louisville, Louisville, KY, USA. 111802-18 week ending
9 SEPTEMBER 2016 PRL 117, 111802 (2016) nnAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. ooAlso at National Research Nuclear University MEPhI, Moscow, National Research Nuclear University MEPhI, Mos ppAlso at Department of Physics, Stanford University, Stanford CA, USA. at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary qqAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physi rrAlso at Flensburg University of Applied Sciences, Flensburg, Germany. ssAlso at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia. tt ssAlso at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia. tt ttAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. 111802-19 111802-19 ttAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. p
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qqAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary.
rr
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https://openalex.org/W4313586190
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https://revistadeliteratura.revistas.csic.es/index.php/revistadeliteratura/article/download/592/600, https://repositorio.uca.edu.ar/bitstream/123456789/16060/1/profec%c3%adas-signos-figuras.pdf
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«Profecías e signos»: las figuras marianas del Antiguo Testamento en Loores de Nuestra Señora de Gonzalo de Berceo
|
Revista de literatura
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Revista de Literatura, 2022, julio-diciembre, vol. LXXXIV, núm. 168
págs. 339-360, ISSN: 0034-849X
https://doi.org/10.3989/revliteratura.2022.02.013
«Profecías e signos»: las figuras marianas del Antiguo
Testamento en Loores de Nuestra Señora
de Gonzalo de Berceo*
«Profecías e Signos»: the Marian Figures of the Old
Testament in Loores de Nuestra Señora
by Gonzalo de Berceo
María Belén Navarro
CONICET / Pontificia Universidad Católica Argentina
mbnavarro@uca.edu.ar
ORCID iD: https://orcid.org/0000-0001-8783-3555
RESUMEN
Loores de Nuestra Señora de Gonzalo de Berceo es un poema cuyo tema es el rol desempeñado por la Virgen María en la economía de la salvación como madre de Dios e intercesora.
Dentro del extenso microtexto narrativo-argumentativo inserto en el macrotexto envolvente de la
plegaria petitorio-laudatoria, se expone entre las cuadernas 4 a 13 una selección de seis figuras
marianas del Antiguo Testamento –la mata de Moisés, el bastón de Aarón, la verga de Jesé, la
alcoba de los salmos, el vellocino de Gedeón y la puerta de Ezequiel–, que anticiparon las cualidades y acciones de María. El presente trabajo se propone estudiar tales figuras con el objetivo
de analizar en cada caso los cuatro sentidos exegéticos –literal, tipológico, tropológico y anagógico– referidos por Berceo y evocar su tradición. Luego se las estudiará como un conjunto para
dilucidar los campos semánticos que pueda haber en común. A partir de ello se verificará la
presencia de un dinamismo semántico-alegórico subyacente, pautado por el macrotexto, que representa las relaciones Dios-María y los hombres en la historia de la salvación. El análisis específico de cada figura y del conjunto a la luz de esta pauta estructural-semántica propuesta posibilitará una mejor delimitación de su intencionalidad, articulación interna con el macrotexto y su
interpretación global.
Palabras Clave: Gonzalo de Berceo; Loores de Nuestra Señora; figura; María; Antiguo
Testamento.
Este artículo es producto del proyecto de investigación «Los Loores de Nuestra Señora, de Gonzalo de Berceo: articulación textual y sentido global», financiado por la Pontificia Universidad Católica Argentina (2014-2018), cuya producción final fue una tesis de
licenciatura (inédita).
*
Copyright: © 2022 CSIC. Este es un artículo de acceso abierto distribuido bajo los términos de una licencia de uso y
distribución Creative Commons Reconocimiento 4.0 Internacional (CC BY 4.0).
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ABSTRACT
Loores de Nuestra Señora by Gonzalo de Berceo is a poem whose topic is the role played
by Virgin Mary in the economy of Salvation as the mother of God and intercessor. Within the
narrative-argumentative microtext inserted into the macrotextual petition-laudatory prayer, there
are six Marian figures of Old Testament between the stanzas 4-13 –the burning bush, the Aaron’s
staff, the branch from Jesse, the chamber of the psalms, the Gideon’s fleece and the door of
Ezekiel–, which predicted her qualities and actions. This paper aims to analyse the four exegetical senses –literal, typological, tropological and anagogical– referred by Berceo in each figure
and to evoke their tradition. Subsequently they will be examined as a set to expose the semantic
fields that they may share. As a result, the presence of an underlying semantic-allegorical dynamism will be verified and it will signify the relationship between God, Mary and men in the
history of Salvation. The specific analysis of the figures and the set considering this proposed
structural-semantic pattern will enable a better definition of their intention, internal coordination
with the macrotext and interpretation.
Key words: Gonzalo de Berceo; Loores de Nuestra Señora; Figure; Mary; Old Testament.
Loores de Nuestra Señora es una obra menor del primer poeta de la literatura en castellano cuyo nombre se conoce, Gonzalo de Berceo, uno de los
principales representantes de la escuela del mester de clerecía. En lo que concierne a su configuración textual, se puede reconocer un macrotexto envolvente1 entre las cuadernas 1 a 4 y 195 a 233 que permiten clasificar al poema como
una plegaria de alabanza-petición2 dirigida a la Virgen María, fundada en el rol
fundamental que desempeña en la economía de la salvación como madre de
Dios e intercesora. Con el fin de ponderar estas cualidades elogiadas, se incluye entre las estrofas 4 a 194 un extenso microtexto (o secuencia incrustrada)
narrativo-argumentativo que esencialmente relata la historia de la salvación
compendiada en María, que funciona como alegato de la loa debida a la Virgen
y fundamento de la petición final (Navarro 2016c).
1 La «presencia mayor» no se determina por cuestiones cuantitativas, sino por ser el
molde discursivo de un texto considerado íntegramente, en otras palabras, el macrodiscurso
(Adam apud Calsamigilia 2012, 253-257).
2 Para esta clasificación textual, nos basamos en un estudio sobre las formas del discurso
religioso en las obras de Gonzalo de Berceo realizado por Javier González, quien define discurso religioso como «todo aquel que postule la existencia real y operante de una dimensión
trascendente o santa a través de su explícita textualización en relación con el locutor, el referente o el alocutario del discurso» (2008, 59). Luego propone una clasificación en cuatro tipos
posibles de macrodiscursos religiosos según el rol desempeñado por la textualización de lo
trascendente-santo: el discurso profético, el discurso-plegaria, el discurso narrativo y el discurso expositivo-instruccional. El discurso-plegaria es aquel donde lo trascendente-santo se
textualiza como alocutario, en segunda persona; lo trascedente se especifica como trascendente-ascendente, en tanto un emisor humano o terreno asciende hacia un receptor divino o celestial: «Yo, hombre, digo a Dios / los ángeles/ los santos» (2008, 65).
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Toda la primera sección, de la cuarta estrofa a la diecinueve, aborda la
Historia de la salvación en el Antiguo Testamento en clave de alegoría
cristiana. El presente trabajo se propone estudiar la selección de figuras
marianas del Antiguo Testamento poetizadas entre las cuadernas 4 a 13 del
poema, con el objetivo de analizar en cada caso los sentidos exegéticos
referidos por Berceo y su tradición. Luego se examinarán las figuras como
un conjunto en pos de dilucidar la funcionalidad de estas alegorías en el
contexto de la obra y los semas que comparten para proponer una sistematización, que planteamos que responde a una dinámica semántico-alegórica
presente en todo el poema, la cual permite plasmar las relaciones divinasmarianas y humanas a lo largo de la historia de la salvación. A partir de
esta sistematización, se podrá delimitar con una perspectiva más integral la
intención, la articulación interna con el macrotexto envolvente y la interpretación de estas figuras marianas.
Figuras marianas del antiguo testamento
1. La presentación de las «profecías e signos» en las cuadernas cuarta y
quinta
Cuand’ engañó la sierpe los parientes primeros /e los sacó de seso con sermones
arteros / de ti s’ temieron luego los falsos lesongeros / mas non fueron del tiempo
nin de hora certeros. // Patriarchas e profetas, todos de ti dissieron,/ ca por Spíritu sancto tu virtut entendieron;/ profecías e signos todos por ti ficieron:/ que cobrarién por ti los qu’ en Adán cayeron (LNS, 4-5).
La primera estrofa del microtexto inicia el racconto de la historia de la
salvación refiriéndose a la tentación mediante el discurso hábil pero embustero
de la serpiente y al pecado del hombre (Gn 3) en los versos 4ab. Inmediatamente en 4c menciona el temor posterior a la enunciación de la promesa mesiánica (Gn 3, 14-15), que Berceo ve realizada en la figura de María, la alocutaria3, rasgo que se conserva del macrotexto. De esta manera, Berceo destaca
Se emplea el término ‘alocutario’, propio de la pragmática del discurso y de la teoría
del diálogo, para discernir entre aquel a quien explícitamente se habla, designándolo mediante marcas discursivas inequívocas como interlocutor y aquel para quien se habla, esto
es, aquel que aun sin ser designado como interlocutor se espera que reciba en última instancia nuestro mensaje –el destinatario– (Stati apud González 2008, 61). Dentro del marco
del cristianismo católico, es evidente que el destinatario último de toda plegaria es Dios,
pero es también posible, conveniente y lícita la plegaria de intercesión, esto es, «aquella
plegaria que toma como alocutario directo de la apelación a un santo en el cielo, a un ángel
o a un ánima del Purgatorio, para que éstos intercedan ante Dios por nuestros pedidos y
lleven hasta Él nuestro arrepentimiento, nuestras gracias o nuestra alabanza» (González
2008, 27).
3
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su rol corredentor, ya presente en el esquema salvífico desde su mismísimo
comienzo.
García de la Concha (1978, 144) considera que este es el primer momento
en el que Berceo introduce textualmente el tiempo como elemento estructural
de la Historia, que vertebra y dinamiza la serie de profecías y signos mesiánicos que seguirán en las cuadernas 5-12. La referencia temporal se concreta en
la proposición incluida adverbial de 4ab y en el adverbio «luego» de 4c, que,
si bien impreciso, indica la posterioridad de la acción, ubicándola ya en la
historia y tiempo de la humanidad.
Respecto a los «falsos lisonjeros» (4c), es una referencia a los falsos profetas, que distorsionan la palabra de Dios por efecto del pecado, interés personal o por la devoción a otros ídolos (Salmos 12,3; 14; Proverbios 7, 21-27).
Tal problemática es abordada especialmente en el Antiguo Testamento, dividiéndose básicamente en dos categorías: quienes hablan en nombre de un ídolo (por ejemplo, los profetas de Baal en 1 Reyes 18, 20-39) y los que pertenecen a la «verdadera religión», pero hablan en nombre de Dios sin cabal
conocimiento o fidelidad al designio (tales como los «profetas-paz», denunciados por Miqueas, Jeremías y Ezequiel (Miqueas 3, 5-8; Jeremías 14, 13-16;
Ezequiel 13) o los profetas cortesanos aduladores (1 Reyes 22, 1-37). La Virgen
María es quien posibilita la discriminación entre los falsos y verdaderos profetas, dado que ‘completa’ la palabra de Dios, mediante su aceptación de su rol
instrumental en la economía de la salvación, acueducto de la encarnación de
Jesús, quien cumple las ‘verdaderas’ profecías. Cabe recordar que el castigo de
los falsos profetas es la muerte y el infierno (Deuteronomio 13, 1-5; Apocalipsis 19, 20; 20, 10).
En contrapunto con el desconocimiento de estos «falsos lesongeros» de 4c,
que no estaban bien informados, tal como ilustra el verso 4d, se presenta la
quinta estrofa. Es un relato sumario en términos narratológicos, dado que en
una estrofa se narra un largo período de tiempo sin detalles de acción, por lo
que el tiempo del relato es menor que el tiempo de la historia4 (Genette 1989,
153). En términos retóricos, funciona como initium narrationis (Lausberg 1966,
I, 271). Se trata de una síntesis de las profecías y signos que se relatarán subsiguientemente y que son, a su vez, la vinculación necesaria entre la caída y la
encarnación desde el punto de vista de la historia de la salvación y también
desde la articulación narrativa interna de LNS, ya que «preanuncian la victoria
de la Virgen sobre el demonio» (Salvador Miguel 1992, 868).
En el verso 5c se presenta la materia narrativa de las siguientes estrofas:
«profecías e signos». Antes de proseguir con el análisis textual es preciso re4 «Propongo [...] llamar historia al significado o contenido narrativo [...], relato propiamente dicho, al significante, enunciado o texto narrativo mismo y narración al acto narrativo productor y, por extensión, al conjunto de la situación real o ficticia en que se produce»
(Genette 1989, 83)
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cordar a qué se refieren estos dos términos en el contexto religioso hispánico
del siglo XIII. Son dos conceptos propios de la religiosidad cristiana medieval
y ligados a tipos discursivos específicos, cuyas características específicas en las
obras de Gonzalo de Berceo ya ha estudiado en profundidad González (2008)
y por ello nos remitiremos a sus definiciones.
Cuando Berceo habla de ‘signos’ se refiere a figura o typus, que es:
Forma o especie de alegoría –in factis o in verbis– por la cual una cosa, un hecho
o una palabra del Antiguo Testamento, en virtud de una analogía o semejanza en
forma o estructura, representa una cosa o hecho del Nuevo Testamento, o bien
una cosa, un hecho o una palabra del Nuevo Testamento representan una cosa o
un hecho predicables del fin de los tiempos, vale decir, una realidad escatológica
(…) La figura funciona, por lo tanto, como una construcción a la vez retórica y
profética (González 2008, 197).
Esta definición reúne las características imprescindibles de la figura, tanto
desde el punto de vista retórico como también teológico. En tanto discurso, es
una forma de alegoría, definida por la retórica clásica como aliud dicitur, aliud
demonstratur, en otras palabras, «metáfora continuada en una frase entera (a
veces más)» (Lausberg 1966, II, 283), en la cual una serie de imágenes refiere
una realidad representada en forma indirecta. Sin embargo, sus componentes
referenciales no son aleatorios, sino que deben aludir inequívocamente a un
objeto histórico de consumación futura y poseer un claro sentido religiosocristiano (Auerbach 1998, 93-115). En consecuencia, el alegorismo se refiere a
«la interpretación espiritual de lo real visible o histórico, considerado como la
imagen o figura de un mundo sobrenatural, lleno de misterios» (Bruyne 1959,
II, 321)5. En un estricto sentido teológico, la alegoría cristiana o in factis corresponde a la interpretación espiritual o profética de ciertos hechos históricos
referidos por la Biblia («sentido literal o histórico»), especialmente del Antiguo
Testamento. El sentido espiritual puede a su vez subdividirse en tres: el «sentido alegórico» o «tipológico», que alude a los hechos del Nuevo Testamento
que dan significado y primera consumación a lo ocurrido en el Antiguo Testamento; el «sentido tropológico o moral», que indica las obras que cada cristiano debe realizar, una norma de conducta diaria según el modelo de tales hechos
del AT y del NT; y el «sentido anagógico» o «escatológico», la segunda y
definitiva consumación que tales hechos han de alcanzar en el fin de los tiem5 «En la visión simbólica, la naturaleza, incluso en sus aspectos más temibles, se convierte en el alfabeto con el que el creador nos habla del orden del mundo, de los bienes
sobrenaturales, de los pasos que hay que dar para orientarnos en el mundo de manera ordenada para adquirir los premios celestes. Las cosas pueden inspirarnos desconfianza en su
desorden, en su caducidad, en su aparecérsenos fundamentalmente hostiles: pero la cosa no
es lo que parece, es signo de otra cosa. La esperanza puede volver, por lo tanto, al mundo
porque el mundo es el discurso que Dios hace al hombre.» (Eco 1997, 70)
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pos (De Lubac 2000, II, 41-50; 83-143; 179-187)6. Por lo tanto, dado este
valor de preanuncio analógico de una verdad arcana y futura, las figuras son
también profecías de dos consumaciones, una neotestamentaria y otra escatológica, definitiva en el fin de los tiempos:
La profecía figural implica la interpretación de un proceso universal y terrenal
por medio de otro; el primer proceso significa el segundo, y éste consuma aquél.
Ambos continúan siendo algo provisional e incompleto, se refieren mutualmente
el uno al otro y señalan hacia un futuro inminente que será el acontecimiento
pleno, real y definitivo (Auerbach 1998, 106).
Si bien toda figura es una profecía, no toda profecía es figura. Por eso es
preciso que también se aborde la definición propuesta por González para ‘profecía’ desde su concepción teológica:
Es el conocimiento, en última instancia inspirado por Dios, de una verdad objetiva oculta al entendimiento natural de uno, varios o todos los hombres, ya por
defecto de la facultad cognitiva humana, ya por incognoscibilidad intrínseca de
la verdad en cuestión […]. La profecía no anticipa la realidad: la profecía instaura y restaura la realidad (González 2008, 41; 43).
Es necesario comentar algunos puntos de esta definición. En primer
lugar, González destaca una cualidad del referente: es una verdad objetiva
que siempre se cumple, en otras palabras, no hay una identificación plena
entre el término ‘profecía’ y ‘predicción del futuro’: no necesariamente debe
manifestar hechos futuros, puede también tratarse de hechos retrospectivos
o actuales que resulten inaccesibles a la inteligencia humana o en cuya
significancia se quiera profundizar (González 2008, 38-39). Asimismo,
cuando la profecía es verbal (y no mental), se configura como una palabra
performativa: hay una identificación entre el decir y el hacer ese algo7; el
6 Un dístico condensa la doctrina de los cuatro sentidos escriturarios: «Littera gesta
docet; quod credas allegoria,/ moralis quid agas, quo tendas anagogia» (apud De Lubac
2000, I, 1)
7 La teoría de los actos de habla según John L. Austin distingue, en cada enunciado
lingüístico, tres actos o modos de «hacer algo» mediante ese enunciado: un acto locutivo,
que consiste en el significado literal generado por una cadena de sonidos (acto fonético)
organizados en palabras construidas según determinada morfología (acto fático) y portadoras
de un sentido y una referencia determinadas (acto rético); al realizar este acto locutivo, el
hablante efectúa asimismo un segundo acto, llamado ilocutivo, que asigna un valor intencional (prometer, ordenar, pedir, advertir, felicitar, sugerir, etc.) a las palabras emitidas; en
tercero y último lugar, existe un acto perlocutivo, que son las consecuencias o efectos que
el enunciado produce sobre los pensamientos, sentimientos o acciones de la audiencia. En
otras palabras, el acto ilocutivo se realiza al decir algo y el perlocutivo se realiza porque se
ha dicho algo. El acto locutivo posee significado, el ilocutivo posee fuerza y el perlocutivo
efectos (Austin 1990, 138-168; Calsamiglia 2012, 10, 186-187).
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hablante –en el caso de las profecías berceanas, normalmente Dios o un
conocedor inspirado que habla por él– emplea el enunciado para realizar
mediante él una acción, en este caso, la instauración de una realidad, porque
la palabra de Dios indefectiblemente hace existir lo que profetiza (González
2008, 43-45).
Para analizar las profecías ya no como concepto teológico sino como una
clase de discurso religioso, González (2008, 41-43; 49-54; 346) propone estudiar distintas categorías en las profecías presentes en la obra de Gonzalo de
Berceo, a saber: el modo de manifestación del acto de conocimiento profético
(verbal o mental); el tipo de discurso verbal (directo o indirecto8); el lugar de
verificación (intratextual, extratextual, combinada, atextual); los alcances de la
verificación (necesaria –en términos absolutos– o condicional –media el libre
albedrío–); la intención del hablante (formal –enunciación voluntaria– o material –enunciación involuntaria–) y la superestructura discursiva (narrativa, exhortativa, descriptiva o mixta).
Estas nociones resultan cardinales para comprender las subsiguientes secciones de LNS, dado que facilitarán el análisis profundo de los sentidos y
alcances de la palabra poética. En lo que respecta puntualmente a esta quinta estrofa, González (2008, 350) la caracteriza discursivamente como una
profecía verbal en estilo indirecto, sin un lugar de verificación textual o extratextual específico, de verificación necesaria, intención formal y superestructura discursiva inespecífica, clasificación con la cual adherimos, dado que,
en primer lugar, es una cuaderna en la cual la voz enunciadora sintetiza la
materia profética que posteriormente se detallará. Los sujetos locutores de
dichos discursos proféticos y signos son los «patriarchas e profetas» (5a),
enunciados en un polisíndeton de pareja inclusiva, pues engloba así a todos
los personajes significativos del Antiguo Testamento; esto se refuerza en el
segundo hemistiquio: «todos de ti dissieron» (5a), repetido luego en paralelismo por 5c: «todos por ti ficieron». Asimismo, se textualiza la fuente que
legitima su conocimiento: el Espíritu Santo (5b) y el referente de los signos
y profecías: «de ti» (5a), «tu virtud» (5b). La finalidad de estos también se
adjudica a María: «por ti» (5c). En consecuencia, el objeto profético es María, sus cualidades y el rol que desempeñaría en la historia de la salvación,
no un hecho puntual, por lo tanto su verificación textual o extratextual no
puede ser ceñida. Nótese, además, que las tres acciones en pretérito perfecto
–«dissieron» (5a), «entendieron» (5b) y «ficieron» (5c)– de esta estrofa, rea8 Genette (1989, 229) diferencia el discurso narrativizado o contado, donde las palabras
quedan asumidas por el narrador y tratadas como un acontecimiento entre otros, del discurso transpuesto o en estilo indirecto, donde la presencia del narrador se nota en la sintaxis de la frase subordinada y no autónomo como cita. El narrador «condensa [las oraciones], las integra en su propio discurso y, por lo tanto, las interpreta en su propio estilo». En
general, en LNS, se utiliza más bien el segundo, pues Berceo suele indicar la fuente.
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lizadas por los profetas, son fundamentales para el proceso de la palabra
profética: primero entendieron la verdad revelada gracias al Espíritu Santo;
luego comunicaron la palabra de Dios y obraron en consecuencia. Por estas
razones, Berceo puede asegurar en tensión proléptica en 5d: «que cobrarién
por ti los qu’ en Adán cayeron».
El verso 5d es la primera mención textual al rol corredentor de la Virgen e
implícitamente alude a la contraposición tradicional Eva-María. Berceo está
retomando la noción de una María intercesora, restituidora del orden trastocado por Eva, tal como explica Carol: «María, como nueva Eva, al representar a
la humanidad cuando consintió en la encarnación y al ofrecer la víctima en el
calvario, reparó el daño causado por Eva» (1964, 36). Subyace en esta dicotomía Eva-María la idea cardinal de una restauración por un proceso de correspondencia invertida: la misma naturaleza que causó la caída debe levantarse,
en otras palabras, a través de una virgen (Carol 1964, 37; 112-115). La tradición
de esta tipología encuentra sus raíces en la patrística occidental –Justino Mártir, Irineo de Lyon, San Jerónimo– y se extiende luego a los doctores de la
Iglesia medievales, como San Bernardo de Claraval, uno de los responsables
de la propagación de la doctrina y adoración marianas y posible fuente inmediata de Berceo.
Se desarrolla luego a manera de amplificatio entre las cuadernas 6 a 12 el
relato anunciado en la quinta estrofa de los signos y profecías, que aluden o
tienen como protagonista directa o indirecta a María. Los seis signos se plasman
como figuras, muy divulgadas en la tradición eclesiástica: cuatro propiamente
marianas y otras dos tradicionalmente cristológicas, pero reinterpretadas por
Berceo (Deyermond 1981, 59). En tanto figuras, son retóricamente alegorías
perfectas, dado que «no es dable encontrar ninguna huella léxica del pensamiento mentado en serio» (Lausberg 1966, II, 285), en otras palabras, Berceo
explica de manera expresa el significado de cada uno de sus componentes,
estableciendo el sentido literal-histórico y el sentido alegórico. De esta manera,
se mantiene una disposición en la distribución de cada estrofa referida a un
único signo (con la excepción de las cuadernas 8-9): primero se enuncia la
figura, que es prospectiva al momento de su enunciación bíblica, con su sentido literal-histórico y luego se la relaciona con los hechos que le dieron cumplimiento en el Nuevo Testamento, tenidos ya por pasados (doblemente, por
los tiempos de su enunciación y por los de su verificación) y como tales mencionados ahora narrativamente mediante verbos en pretérito (González 2008,
160). Si bien este texto de Berceo no es bíblico ni de inspiración divina, sí se
configura como hipertexto bíblico, incluyendo imágenes, ideas y significados
que proceden directa o indirectamente de la Sagrada Escritura, por lo tanto es
pasible de ser interpretado como alegoría cristiana o in factis. En consecuencia,
no sólo es preciso analizar los sentidos histórico y alegórico explícitos, sino
también indagar e interpretar los dos sentidos espirituales restantes de la exéRevista de Literatura, 2022, vol. LXXXIV, n.º 168, 339-360, ISSN: 0034-849X
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gesis (tropológico y anagógico) que haya podido heredar lícitamente de la
fuente bíblica9.
2. La mata encendida de la sexta estrofa
La mata que paresco al pastor encendida/ e remanesció sana com’ ante tan cumplida,/ a ti significava, que non fust’ corrompida/ nin de la firmedumbre del tu
voto movida. (LNS, 6).
Se refiere a la zarza10 que arde pero no se consume de Éxodo 3,2, a través
de la cual se apareció a Moisés el Ángel de Dios; esta visión posibilita el encuentro con Dios, que se revela ante el profeta otorgándole su Nombre. La
combustión espontánea de la zarza es un hecho relativamente frecuente en el
desierto; la maravilla del signo radica más bien en ser un fuego que no consume: es un medio de la teofanía. Es la señal de la visita del Dios vivo, la presencia de la santidad divina, en su doble aspecto: atractivo y temeroso (LéonDufour 1965, 307). El sentido alegórico deriva del primero, dado que es una
llama iluminadora, purificadora y fecundadora, según lo explica Berceo en los
últimos versos de la estrofa (6cd). Menéndez Peláez (1981, 124) sostiene que
se trata de una imagen común con San Bernardo: «Magna plane visio, rubus
ardens sine combustione, magne signum, mulier illaesa manens amicta sole»
(PL 183, 432). De la misma manera que el Ángel de Dios se presenta como
fuego en la mata sin causar daño, tampoco saldrá menoscabada María al recibir a Cristo11. Para Menéndez Peláez (1981, 124) y para Deyermond (1981,
162) se refiere a la virginidad de María mientras que García de la Concha (1992,
66) y Ruiz Domínguez (1990, 162) la relacionan también con la concepción
9 «Cuando los poetas se inspiran en la Escritura y aun sencillamente en la Naturaleza,
puesto que en la estructura de los hechos históricos y físicos Dios ha inscrito la figura de
realidades sobrenaturales y misteriosas, ¿no es de suponer que en todas las obras haya al
menos un germen de significación alegórica...?» (Bruyne 1959, II, 348). Bruyne defiende
cierto alegorismo universal y matiza de esta manera las afirmaciones de Santo Tomás de
Aquino («unde in nulla scientia, humana industria inventa, proprie loquendo, potest inveniri nisi lirteralis sensus» (Quod. VII, 6, 16) y «Fictiones poeticae non sunt ad aliud ordinatae nisi ad significandum [y su Sigmficado] non supergreditur modum litteralem» (Quod.
VII, 6, 16, ob. 1, y ad 1), a través de las cuales limitaba la poesía mundana al sentido literal,
aunque hubiera una figura retórica (alegoría in verbis) con un sentido parabólico.
10 La zarza es una maleza de arbustos bajos, ramosos, frecuentemente adornada de
espinas (que simboliza el fruto de la maldición de la tierra); por lo cual, tradicionalmente
es una figura cristológica que refiere a la corona de espinas de la Pasión; la corona de un
rey, humillante y dolorosa, verdadero símbolo de todo el sufrimiento y también de todas las
glorias de la redención (Vigouroux 1922, 1087-1088; 1895-1896).
11 En gran medida, se asemeja a la imagen del rayo de sol (209ab) y el fuego templado
de los niños (212bc) de la conclusión del macrotexto.
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inmaculada. Por nuestra parte, resulta evidente que el verso 6c se refiere a la
concepción inmaculada: María se encuentra preservada de la mancha del pecado original (Carol 1964, 26) mientras que 6d alude a su voto de castidad,
mantenido antes, durante y después del parto de Jesús. La explicación del
sentido neotestamentario aplicado a María adopta una estructura descriptiva: el
verbo entitativo ‘ser’ expreso y elidido, auxiliar en dos frases verbales pasivas.
En 6c expresa un estado permanente –la incorruptibilidad de María– y en 6d
una acción constante e iterativa –no ser movida de su voto–. Nótese, además,
que se la caracteriza mediante la negación: «non fust’ corrompida» (6c) y «nin
de la firmedumbre del tu voto movida» (6d).
El fuego indica la presencia divina, pero es también la exigencia de pureza
del Dios santo: sólo el elegido de Dios comprueba que ha podido afrontar su
presencia sin morir (Léon-Dufour 1965, 307-309), tal como pudo Moisés (sentido histórico) y como María (sentido alegórico). En consecuencia, el sentido
tropológico es la respuesta humana que debe ser dada a la exigencia de la
pureza en la vida del cristiano, mediante la recepción del fuego de Dios, su
Amor, a través de la Palabra, que arde en su corazón sin quemar y purificándolo. Por último, el sentido escatológico se encuentra en la interpretación del
fuego-devorador como signo de la intransigencia de Dios frente al pecado:
devora al que encuentra, de la misma manera Dios respecto al pecador endurecido, al réprobo que encontrará su lugar en el Infierno (Vigouroux 1922,
2225-2227). No sucede lo mismo con sus elegidos, los justos, transformados
por entrar en contacto con él (y en este sentido, también se podría asociar intra
y extratextualmente a las leguas de fuego del Espíritu Santo en Pentecostés,
referido en las cuadernas 155-159).
3. El bastón de Aarón de la estrofa séptima
A ti catava, Madre, el signo del bastón/ que partió la comanda que fue por Aarón/
fust’ sin raíz e seco adusso criazón/ e Tú pariste, Virgo, sin toda lessïón (LNS, 7).
Su fuente es Números 17, 16-23. El báculo de Aarón, el hermano de Moisés, floreció en almendras12 como signo inequívoco de la voluntad de Dios de
12
Esta misma figura del bastón de Aarón aparece en el prólogo de Milagros de Nuestra
Señora de manera duplicada: en los versos 39ab «Es dicha vid, es uva, almendra, malgranada,/ que de granos de gracia está toda calcada» y en la estrofa 41: «Si metiéremos mientes en ello otro bastón/ que partió la contienda que fue por Aärón,/ ál non significava, como
diz la lectión,/ si non a la Gloriosa, esto bien con razón». La segunda estrofa es la más
cercana a Loores en cuanto a su semántica, ya que alude a la función del signo de revelar
la voluntad divina, su elección (7ab), pero el poema que nos compete se explaya un poco
más en la explicación alegórica al referir la sequedad, las condiciones aparentemente inhabilitantes para el acontecimiento de la concepción. Por el contrario, la primera estrofa seña-
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que aquél fuera consagrado sumo sacerdote. Según Deyermond (1981, 59), no
era un signo referido a María tradicionalmente, sino a Cristo, pero no lo consigna así Carol (1964, 76). Berceo explica la alegoría en la segunda parte de
la estrofa (7cd); se refiere a la inesperada fertilidad de María al mismo tiempo
que destaca en contrapunto su virginidad (subrayada por el vocativo en 7d), a
pesar de su maternidad, manifestada reiteradamente por el verbo activo «pariste» (7d) y el vocativo «Madre» (7a). Aparece nuevamente la caracterización
por la negación: «sin raíz e seco» (7c) y «sin toda lessïón» (7d).
Como signo, esta figura pertenece a una dimensión de apertura; la fe de
María, esbozada ya en la figura anterior de la zarza, es ahora fecundidad espiritual; en tanto madre, se enfatiza el acto donativo del poder milagroso hacia
el prójimo para su salvación. Tropológicamente alude a la vida cristiana que
encuentra su «florecimiento» en el servicio a Dios, mediante la aceptación y
realización de Su voluntad, representada por el bastón. En consecuencia, su
sentido escatológico es la salvación del Pueblo de Dios.
4. La vara de Jesé en las cuadernas octava y novena
En ti s’ cumplió, Señora, el dicho d’ Isaía:/ que de radiz de Yesse una verga saldría/
e flor qual non fue vista dend’ se levantaría,/ Spíritu con siet’ dones en la flor
posaría.// Madre, Tú fust’ la verga, el tu fijo la flor/ que resucita muertos con su
suave odor,/ saludable por la vista, vidable por savor/ pleno de los siet’ dones,
sólo d’ ellos dador (LNS, 8-9).
Esta figura tiene su fuente en Isaías (11, 1-3); tradicionalmente no se refería a María, sino a Cristo, anunciando la pertenencia del Mesías al linaje de
David, hijo de Jesé (Deyermond 1981, 59). Pero Menéndez Pélaez (1981, 124)
la identifica como imagen mariana común de Berceo y San Bernardo (PL 182,
1144). Discursivamente ambas estrofas se constituyen como una profecía verbal
de superestrutura narrativa (González 2008, 350). Primero, en la estrofa octava,
Berceo recupera sintéticamente en estilo indirecto la profecía de Isaías, constituyéndose como relato singulativo –al contar en un enunciado único lo que
ocurrirá una vez (Genette 1989, 173) – y anacrónico –al existir una discordancia entre el orden de la historia y del relato (Genette 1989, 92) –. En tanto lo
referido por Isaías evoca por adelantado un acontecimiento que será relatado
la las propiedades del fruto (en Loores solamente flor), la almendra, de un gran valor simbólico: González explica que en la lengua hebrea existe una identidad fónica entre
‘almendra’ y ‘el que vela’ –shaqued–, de donde la mención de Jeremías de una virga vigilans
(Jer. 1, 11), a partir de lo cual expone: «ambos contenidos, la fecundidad repentina y milagrosa de la vara de Aarón y la condición vigilante y siempre atenta de la almendra, fueron
tomados luego como figuras de María, igualmente fértil contra toda ley natural y alerta y
dispuesta ante la anunciación del Ángel; como dato adicional, la almendra florece tempranamente, aún en invierno, y el nacimiento virginal de Jesús fue en diciembre» (2013, 125).
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posteriormente, es narrativamente una prolepsis interna repetitiva (Genette
1989, 95).
Luego la estrofa novena expone el sentido alegórico neotestamentario, aludiendo a cualidades permanentes a través del verbo «ser» (9a) y adjetivos (9cd),
salvo por una sola acción: «resucita» (9b) de índole iterativa. El árbol de Jesé
es símbolo de las generaciones, cuya historia sintetizan las Sagradas Escrituras
y que culmina con la Virgen y Cristo. De la raíz (entendida como el origen de
la posterioridad) del árbol de Jesé –padre del rey David– nacería un vástago
destinado a reinar sobre todos los judíos y en quien se posaría el Espíritu Santo con sus siete dones (8d; Isaías 11, 2-3). Esta flor que saldría de la rama es
Jesús. Su madre María es la rama de la que nace este brote (9a). De esta manera, la verga «significa a la Virgen María, la nueva Eva, que ha concebido por
mediación de la gracia» (Ruiz Domínguez 1999, 164).
En la estrofa novena la figura adquiere un verdadero talante poético. La flor
posee un sentido plenamente simbólico-alegórico, que se profundiza al enumerar sus cualidades sensoriales agradables: el suave aroma, poder milagroso con
el cual resucitará muertos (9b); la vista, que brinda salud (figurativamente, la
salvación) y sabor, que es «vidable» (9c), en otras palabras, grato, pero literalmente «que da vida» (Salvador Miguel 1992, 870). Vuelve a resaltar luego los
siete dones de la flor (9d) en correspondencia con lo ya anunciado en 8d.
En consecuencia, tropológicamente se puede interpretar que la rama que da
vida (la flor) es aquella alma cristiana que reconoce a Dios y puede cultivar la
virtud, particularmente la caridad en cuanto amor a Cristo y al prójimo, lo cual
le permite gozar de los frutos de la vida espiritual. Su contrapartida serán las
ramas sin fruto (Mateo 7:15-20 y Marcos 4: 14, 18-19), aquellos en los que la
Palabra de Dios no es admitida, como los judíos incrédulos, que serán retratados en 15cd y 17d13. Escatológicamente se trata de la vida plena en el Paraíso
Celestial (Vigouroux 1922, 2287-2288).
5. El signo de la «cambariella» en la estrofa décima
Tú fust’ la cambariella que dize el psalmista,/ end’ salió el esposo con la fermosa
vista,/ gigant’ de grandes nuevas que fizo grand conquista,/ rëy fue e obispo e
sabidor legista (LNS, 10).
Se refiere al tálamo del Salterio (19, 6): «Soli posuit tabernaculum in eis,et
ipse, tamquam sponsus procedens de thalamo suo, exsultavit ut gigas ad cu13 Ruiz Domínguez (1999, 164) estipula que la verga también podría aplicarse a la
Iglesia Universal, descendiente de María y Cristo. Esta interpretación no es incompatible
con la propuesta aquí consignada, dado que la vida cristiana es necesariamente un llamado a vivir en tal institución humano-divina, en la que el hombre puede hallar la luz, el
perdón y la gracia.
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rrendam viam». Berceo explica que María es la alcoba del salmo, de la cual
sale el esposo (10b), muy similar a la imagen del verso 199b de la conclusión:
«Tú fuisti reliquiario pleno de sanctidat». Ambas imágenes designan al seno de
María, donde se guardó y cuidó a Jesús. La voz enunciadora prosigue luego a
ampliar la figura, indicando las cualidades de éste: primero nuevamente la
sensorial, pues cuenta «con la fermosa vista» (10b) para luego avanzar en sus
roles ejercidos: «gigant’ de grandes nuevas que fizo grant conquista,/ rëy fue e
obispo e sabidor legista» (10cd). Discursivamente es una profecía verbal de
estilo indirecto de superestrutura narrativa (González 2008, 350), ya que la
acción anticipada en la prolepsis de 10b se relata singulativamente. Pero luego
el sentido alegórico principal se torna más bien descriptivo al emplear verbo
entitativo ‘ser’ y adjetivos que refieren a características (10d), salvo por la
referencia a una acción puntual «fizo grant conquista» (10c).
El nombre de esposo es uno de los que se da tradicionalmente a Dios y que
expresa su amor a su criatura y su pueblo; es gratuito y fiel, insondable y eterno, triunfará y transformará a la infiel en una esposa virginal, con la que se
unirá mediante una alianza eterna14. El sentido alegórico de tal esposa, resultante de la nueva alianza, es la Iglesia. En consecuencia, tropológicamente se
trata de la participación del cristiano de la vida trinitaria, de unirse con el Hijo
de Dios para ser hijo del Padre celestial (Léon-Dufour 1965, 264-266). Para
ello, se puede sintetizar el sentido tropológico como la batalla diaria del cristiano contra el demonio, la carne y el mundo, siempre victoriosa en la unión
con Cristo. Escatológicamente se refiere a la unión de Cristo con la humanidad
regenerada, con la Iglesia Triunfante, cuyo resultado es la Felicidad Celestial
(Vigouroux 1922, 759-773).
6. El vellocino de Gedeón de la estrofa undécima
La tu figura, Madre, traïé el vellocino/ en qui nuevo miraglo por Gedeón avino;/
en essi vino pluvia, en ti el rey divino;/ por vencer batalla, Tú abrist el camino
(LNS, 11).
La figura se refiere a Jueces 6, 36-40. Se narra cómo Gedeón solicitó a Dios
una señal para saber si él era el elegido para guiar a Israel a la victoria en la
batalla: echaría sobre el suelo el vellocino toda la noche y, si al amanecer lo
encontraba empapado en rocío, mientras el suelo permanecía seco, sabría que
él era el predestinado; así se hizo. Es otra imagen compartida con San Bernardo señalada por Menéndez Peláez (1981, 124). En este caso, esta figura profética alude a la concepción virginal de María, por la milagrosa virtud de generar
rocío en medio de la sequedad. Esta interpretación está acentuada por el voca14 Cfr. Isaías 54, 5; Jeremías 3, 20; Oseas 1,2; 2, 21-22; 3,1; Efesios 5, 21-33; Mateo
9, 15; Apocalipsis 21, 9-10.
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tivo «Madre» (11a). La lluvia o rocío (en la fuente bíblica), a su vez, remite a
la prosperidad, como fuente de gracia y de vida (Vigouroux 1922, 1208-1210).
Ruiz Domínguez señala que también existe una relación analógica entre María
y Gedeón en lo que compete a su relación con Dios, según el siguiente esquema: «comunicación del mandato divino, objeción del elegido, refutación de la
objeción mediante la promesa de Yavé y señal que confirma el origen del mandato» (1999, 163).
Por otro lado, esta misma imagen es utilizada en Milagros de Nuestra
Señora: «Ella es vellocino que fue de Gedeón,/ en qui vino la pluvia, una
grand vissïón» (34ab), pero con ciertos matices distintivos. Para García de la
Concha (1978, 146), Berceo agrega dos datos enriquecedores en la explicación de la alegoría en Loores: por una parte, la referencia en el verso 11c:
«en essi vino pluvia, en ti el rey divino» recapitularía la tradición de los
profetas menores (Os. 14,6; Zach 8,12), compendiada luego en el himno litúrgico del Adviento, que asigna a la lluvia sobre el vellocino el valor de
signo del Mesías Salvador. Por otra parte, en 11d, «por vencer la batalla, Tú
abrist el camino», la imagen encuentra su contexto bélico histórico reinterpretado por el sentido espiritual: la batalla libertadora del Pueblo, que Gedeón
va a emprender, pero quien verdaderamente la inicia es María, al aceptar ser
madre de Jesús, en la batalla contra el pecado y por la salvación, diariamente en lo que refiere a la tropología y al final de los tiempos como consumación
en su sentido escatológico.
7. La puerta cerrada del Templo en estrofa duodécima
La puerta bien cerrada que dice Ezechiel,/ a ti significava que siempre fuisti
fiel;/ por ti passó señero el Señor d’Israel/ e d’esto es testigo el ángel Gabriel
(LNS, 12).
Su fuente es Ezequiel (44, 1-3). Es una imagen común con San Bernardo
(Menéndez Peláez 1981, 125; PL 182, 145). Discursivamente es una profecía
verbal de estilo indirecto de superestrutura narrativa (González 2008, 350). En
cuanto anacronía, es una prolepsis de índole interna repetitiva, ya que alude a
un acontecimiento que posteriormente se contará en detalle (Genette 1989, 124)
en las cuadernas 20-26.
Berceo explica el sentido alegórico aplicado a María describiéndola de la
siguiente manera: «a ti significava que siempre fuisti fiel» (12b). Se refiere no
sólo al parto virginal, sino también a la virginidad (prepartum y postpartum)
de María. Esta puerta siempre permanecerá cerrada porque el Señor ha entrado
por ella, lo cual constituye una acción singulativa. La virginidad se configura
como signo de su fidelidad en un amor exclusivo para Dios y fruto de una
pureza total (Léon-Dufour 1965: 840-842). Por lo tanto, el sentido tropológico
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radica en la fidelidad, esto es, la observancia de la fe y la obediencia a Dios
como eje de la vida cristiana.
El sentido escatológico difiere del enunciado para el signo de la puerta en
Milagros de Nuestra Señora: «Ella es dicha puerta en sí bien encerrada, por
nós es abierta por darnos entrada» (36ab). Esta idea de ‘apertura’, ausente en
Loores, se asocia con la puerta de Jerusalén celestial, que impide la entrada del
enemigo y convierte a María en un medio de entrada a la bendición (Vigouroux
1922, V, 548-555). Sin embargo, en Loores, el foco se encuentra en la cerrazón
con el objetivo de realzar el voto de castidad y virtud de María. En consecuencia, el sentido escatológico de esta puerta cerrada radica justamente en la negativa que representa frente a la tentación y corrupción del pecado. Quien así
permanezca, podrá unirse y participar totalmente en el misterio de Jesús.
Luego de enumerar y explicar estos signos y profecías marianos, se presenta la conclusio en la estrofa decimotercera15, reforzando la verdad y verificación
de estos anuncios en la proclamación de la dignidad de los sujetos locutores y
en la universalidad especial del mensaje. Recapitula con cierta simetría en 13d:
«todos en tu materia salieron verdaderos» el inicio de toda la serie en 5a: «Patriarchas e prophetas todos de ti dissieron», fortaleciendo la verificación histórica de lo dicho y la cohesión de toda la sección.
Interpretación global de las figuras marianas elegidas por Berceo
Finalizado el análisis individual de las figuras, es conveniente proseguir con
un examen global, entendiéndolas como un conjunto integral con una posible
estructura semántico-alegórica subyacente que permita interpretar con mayor
precisión su funcionalidad. Por lo tanto, es preciso identificar los campos semánticos presentes en estas cuadernas para sistematizarlos.
Primero, las figuras se encuentran vinculadas a través del campo semántico
del ‘signo’ y la ‘profecía’ (5c): «significava» (6c, 12b), «catava» (7a), «figura»
(11a), «dissieron» (5a), «dicho» (8a), «dize» (10a, 12a), «mensageros» (13a).
Respecto a esta simetría de los núcleos semánticos, García de la Concha (1978,
148) sostiene que la sección en su totalidad responde a un esquema de ‘signo
+ significado’, articulado sobre el eje del ‘tú’ referencial como alabanza litánica y como planteamiento dialéctico de las variadas formas del anuncio mesiánico que urgen una respuesta. El eje ‘tú’ referencial apuntado por García de la
Concha se verifica no sólo en la semántica de las figuras, sino también en el
aspecto gramatical, dado que prevalecen las formas pronominales que manifiestan el alocutario en segunda persona singular, con sus variantes funcionales:
«Éstos fueron e otros, Madre, tus mensageros,/ muchos ovieron éstos de tales compañeros;/ de todas gentes fueron, ca non unos señeros,/ todos en tu materia salieron verdaderos» (LNS, 13).
15
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sujeto «Tú» (7d, 9a, 10a); posesivo «tu/tus» (5b, 6d, 11a, 13a,d) y término: «de
ti» (4c, 5a, 5c, 5d, 12c), «a ti» (6c, 7a, 12b) y «en ti» (8a, 11c). Estas formas
pronominales se acompañan de vocativos: «Madre» (7a, 9a, 11a, 13a), «Virgo»
(7d) y «Señora» (8a). También son abundantes las formas verbales en segunda
persona: «fust’» (6c, 9a, 10a, 12b), «parist’» (7d) y «abrist’» (11d). El resto de
los verbos adoptan la tercera persona singular, en general para referirse al acto
de habla de los profetas «dice Eccechiel» (12a) o para describir el signo «la
mata que paresco» (6a) y relacionarlo con María «a ti significava» (6c). Luego
se encuentran aquellas que aluden a Cristo a través de la antonomasia16: «end’
salió el esposo con fermosa vista / gigant’ de grandes nuevas que fizo grant
conquista, / rëy fue e obispo e sabidor legista» (10bd), «en essi vino pluvia, en
ti el rey divino» (11c) y «por ti passó señero el Señor d’ Israel» (12c).
Estas seis figuras –la mata de Moisés, el bastón de Aarón, la verga de Jesé,
la alcoba de los salmos, el vellocino de Gedeón y la puerta de Ezequiel– representan cualidades de María, que se pueden compendiar en dos campos semánticos: la ‘clausura’/’pasividad’ y la ‘apertura’/’donación’. El primer eje de
la ‘clausura’/’pasividad’ se ve realizado en las formas verbales pasivas de 6cd
y 8d o acciones en las cuales María es receptáculo o medio de la acción, pero
no agente, indicado por los giros circunstanciales como en «en essi vino pluvia,
en ti el rey divino» (11c), «por ti passó señero el Señor d’Israel» (12c). Se
visualiza también en las formas negativas de 6cd, 7cd y en semas de clausura,
como 7c («seco»), 7d («virgo»), 10ª («cambariella») y 12ª («puerta bien cerrada»), y de lejanía en cuanto a su jerarquía: 8ª («Señora»).
El segundo eje de la ‘apertura’ se manifiesta en formas verbales activas:
«pariste» (7d), «una verga saldría» (8b), «abriste» (11d), y en los vocativos de
7ª, 9ª y 11ª («Madre»). El producto de su acción donativa es Cristo, en quien
se concentran los restantes rasgos: agente de verbos activos («resucita» –9b–,
«salió el esposo» –10b–, «fizo grant conquista» –10c–, «pasó señero el Señor
d’ Israel» –12c–) y en los semas relacionados con los sentidos, que se expanden:
«… con su suave olor / saludable por vista, vidable por abor,/ pleno de los siet’
dones, sólo d’ ellos dador» (9bcd).
Lo que podría considerarse una relación antitética entre los dos conjuntos
semánticos de ‘clausura’ y ‘apertura’ se torna superadora en la figura de Cristo,
referido como la flor, el esposo saliente, el rey, el legista, el obispo y el conquistador, en quien convergen clausura y apertura, pasividad y actividad. Existe entonces en estas cuadernas una dinámica de contraposición de dos fuerzas antagónicas (en este caso, dos campos semánticos) que hallan el equilibrio en la
recíproca contención y neutralización (el tercer campo semántico de la fertilidad
o el fruto crístico). Es una pauta estructural de carácter semántico para plasmar
Existe otra referencia a Cristo por antonomasia en la sección, pero no cuenta con
forma verbal por estar sobreentendida: «Madre, Tú fust’ la verga, el tu fijo la flor» (9a). Es
la primera textualización en tercera persona de Cristo en LNS.
16
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las relaciones humanas y divinas a lo largo de todo el poema de Loores de Nuestra Señora, pero nos compete concentrarnos en las cuadernas 4 a 13.
Esta dinámica textual de contraposición neutralizada de fuerzas había sido
dilucidada y analizada previamente en el prólogo de otra obra de Berceo, Milagros de Nuestra Señora. Por Aquilino Suárez Pallasá (1989) como por González (2013). En primer lugar, Suárez Pallasá (1989) propone como principio
rector del prólogo una dinámica bimembración antitética a partir del estudio de
un grupo de lexemas presentes en el texto –tempestad, tiemplo, temprado– que
remiten a la raíz indoeuropea temp- ‘manifestación de una fuerza en movimiento de extensión-intensión antagónico y contenido’. El trabajo de este crítico le
sirve de punto de partida a González (2013, 21) para postular en tal prólogo
un esquema estructural aún más complejo de oposición ortogonal, conforme a
una organización espacio-direccional de dos estructuras octogonales que incluye como elementos constitutivos la horizontalidad, la verticalidad, lo descendente, lo ascendente, lo centrípeto y lo centrífugo organizado en dos planos: el
divino-mariano (Dios omnipotente y su delegada María) y el humano (los
alocutarios terrenos del poeta y el poeta mismo).
Según este análisis, existen dos ejes constitutivos análogos en ambos planos: la verticalidad y la horizontalidad. En el plano divino-mariano, la verticalidad se identifica con el poder divino (en María delegado o participado), correspondiente al vasallaje exigido por Dios y expresado en la asimetría de
planos y la subordinación de lo terreno a lo celestial. El eje horizontal se identifica con la amistad de Dios para con los hombres, a la misericordia, a la
igualación de lo divino y lo humano en la encarnación. En el segundo esquema,
remitido al plano humano, el eje horizontal corresponde, en el caso del prólogo de Milagros de Nuestra Señora, a la romería en que consiste la vida humana y que tiende a Dios como meta; el eje vertical se identifica con el servicio
prestado a Dios o a la Virgen (la asimilación con las aves que cantan las alabanzas en el ejemplo de Milagros).
Sin embargo, estos dos ejes análogos difieren en la definición de su direccionalidad, dado que en el esquema divino-mariano la verticalidad resulta descendente y la horizontalidad centrífuga con respecto al punto de articulación
de los ejes horizontal-vertical; en tanto en el esquema humano, el eje horizontal es centrípeto, de la periferia al centro (como el romero hacia el árbol del
prado), y el vertical ascendente (como el ave que trepa al árbol). De esta manera, la dinámica de la relación divino-humana en el prólogo de Milagros de
Nuestra Señora queda plasmada en un doble esquema (divino y humano) de
cuatro ejes: horizontal-centrípeto, vertical-descendente, horizontal-centrífugo y
vertical-ascendente (González 2013, 29-33).
El esquema semántico-alegórico de González de dos planos (divino y humano) de cuatro ejes (horizontal-centrípeto, vertical-descendente, horizontalcentrífugo y vertical-ascendente) es viable y conveniente también para Loores
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maría belén navarro
María y los hombres, aunque con rasgos peculiares vinculados con la especificidad textual del microtexto en el cual se encuentran insertos.
En este fragmento de las cuadernas 5-13, por ser un discurso explicativo
de figuras teológicas, sólo tenemos el plano divino-mariano; no se textualizan
agentes humanos, por lo cual hay dos direccionalidades no textualizadas: lo
ascendente y lo centrípeto, que sí se encuentran en otras secuencias narrativas
posteriores del poema. La direccionalidad vertical-descendente es la que prevalece a nivel textual, tanto para el plano propiamente divino, en tanto expresión de la omnipotencia celestial que desciende a la tierra como contenido de
fe (González 2013, 36), ya en las manifestaciones concretas de Dios en signos
que exhiben su poder (el bastón, la zarza, el vellocino), ya en las profecías
verbales que prefiguran la Encarnación (el árbol de Jesé, el esposo de la cambariella, la puerta). Además, la ‘clausura’ propia de la virginidad mariana se
identifica con el eje vertical-descendente por significar el poder, la lejanía.
En tanto María acepta el designio de ser la Madre de Dios, su ‘clausura’ se
convierte en paradójica ‘apertura’, que en sí misma pertenece al eje horizontal
centrífugo: la misericordia, la cercanía. Sin embargo, en estos signos se combina simultáneamente con el eje de poder-clausura descripto anteriormente, por
lo cual resulta un equilibrio armónico y la potenciación recíproca en un campo
mixto de poder-misericordia, verticalidad-horizontalidad, que encuentra su eje
de conjunción en la idea de fertilidad, entendida a la vez como expresión de
poder en el acto de concepción y generación, y de misericordia en el acto de
parición y donación al prójimo del fruto, que posibilita la vida total y plena
(González 2013, 130-132). La flor de este campo mixto es Cristo, que los integra y potencia. Esta coincidencia de direccionalidades se corresponde con la
doctrina de la intercesión mariana; un esquema triádico en el que operan dos
mediados y un mediante:
La marca personal y física de la condición intercesora de María entendida como
oxímoron, como paradoja y coincidentia oppositorum donde toda contradicción
se resuelve en una lógica superior y mistérica es, claro está, la coexistencia de la
virginidad y la maternidad (González 2013, 132).
María es necesaria intercesora entre la divinidad y la humanidad, por ser
humana exclusiva y plena e inmaculada y perfecta; necesario instrumento para
la encarnación y salvación; pero el verdadero y único objeto de fe es Cristo,
núcleo de revelación definitiva que ofrece el Nuevo Testamento y que Berceo
procederá a relatar en las estrofas 20-169. Se alaba a María por ser la ‘puerta’
que nos permitió en la historia de la salvación acceder a Cristo, a cuya adoración todo se subordina.
Al considerar la profundidad que adquieren los signos a la luz de este esquema, la teoría de la intencionalidad de toda la secuencia propuesta por García de la Concha (1978, 146) pierde rigor. El crítico considera que los signos
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y profecías marianos de las cuadernas 5-13 cobran mayor desarrollo imaginativo al servicio de una intención catequística. Si bien la índole textual del
fragmento es mayoritariamente explicativa, esto se debe al contenido teológicofigural que necesariamente requiere esa superestructura por ser una alegoría
perfecta. Lo verdaderamente esencial son las figuras seleccionadas: es evidente que más bien se está intentando ensalzar a María por su fidelidad, por su
incorruptibilidad, pero sobre todo por su rol intermediario, su rol instrumental
en la historia de la salvación. Cierto didactismo o ejemplaridad didáctica radica en Loores sólo como un efecto perlocutivo secundario, en otras palabras,
como consecuencias y efectos concretos y reales en los receptores, que escapan
a las intenciones primarias del emisor, puesto que el discurso de alabanza puede resultar contagioso o formativo para algún receptor individual y puede ser
tenido por imitable, por lo cual deviene secundaria y ocasionalmente en ejemplar y didáctico (González 2013, 157).
Esto coincide con nuestra hipótesis de trabajo17 acerca de estos microtextos
–tanto los narrativos como los explicativos y los directivos– como narrationesargumentationes subordinadas a la intención laudatoria y petitoria, cuyo objeto
es ratificar o reforzar el elogio del macrotexto de la plegaria, como un caso de
amplificatio, fenómeno propio de la ornatus. (González 2008, 160; Navarro
2016c). Tal como indica Lausberg en lo que concierne a las estructuras retóricas, «a lo largo de la narratio se van sembrando puntos de apoyo que utilizará
después la probatio» (1966, I, 284). Se trata de aumentar la fuerza de una idea
indudable y ya afirmada como res certa (el poder, la misericordia y la laudabilidad de María) mediante la acumulación tanto de res (los hechos que dan
cuenta de su poder, misericordia y laudabilidad a lo largo de la historia de la
salvación) como de verba (la organización discursiva de esos hechos mediante
técnicas narrativas y retóricas).
17
No es el objeto del presente trabajo, pero está implicado en una hipótesis mayor de
investigación sobre la interpretación de Loores de Nuestra Señora. La finalidad de la obra
ha sido debatida –especialmente Dutton (1980), Menéndez Peláez (1981), García de la
Concha (1978; 1992), Ruíz Domínguez (1990), González (2008)–, con dos posturas básicas
predominantes: la latréutica y la catequística. Si bien es cierto que un propósito moralizador
no es necesariamente incompatible con una finalidad latréutica, es conveniente discernir cuál
de los dos resulta el objeto último y global de la obra. La hipótesis basal de nuestra investigación ha sido la finalidad global latréutica, secundariamente catequística, establecida por
el macrotexto de la obra, que envuelve y brinda unidad a los microtextos insertos. Para ello
se ha abordado el género, la macroestructura y superestructura de la obra (Navarro 2016c)
en pos de limitar su acto de habla fundante. También se ha analizado el rol de los alocutarios
humanos en los microtextos (Navarro 2016b), la heterogeneidad de los enunciados expositivos (Navarro 2017b), la función de la meditación de la Cruz (Navarro 2016a), los enunciados directivos de la escatología (Navarro 2017a), entre otras líneas de análisis de la investigación todavía no publicadas en prensa, pero presentes en nuestra tesis de licenciatura.
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maría belén navarro
Conclusiones
Esta primera sección del poema (4-19) demuestra el dinamismo frecuente
de la lectura del Antiguo Testamento durante la Edad Media mediante la interpretación alegórica cristiana. Berceo expone en sus versos el sentido literal (la
‘letra’, como él mismo la designa en 15d) y el alegórico, prospectivo al momento de su enunciación bíblica pero retrospectivo al momento de la enunciación del poema. El Antiguo Testamento funciona entonces primero en sí mismo
y luego en relación preparatoria con respecto al Nuevo, que es la última palabra, por ser definitiva y eterna. El espíritu de la letra es Cristo, por lo cual es
Él quien brinda unidad a la Escritura, porque es su consumación y su cumplimiento (De Lubac 2000, I, 225-267) y en quien hallan ambos Testamentos su
interdependencia indisoluble, por ser el centro de la historia de la salvación.
En consecuencia, María es ensalzada desde su rol de causa instrumental en lo
que concierne a la Encarnación: su maternidad virgen y su fidelidad a Dios, lo
cual justifica su condición de intercesora.
Ya en este primer momento de la narratio se puede observar una disposición compositiva que se desarrollará a lo largo del poema: el signo divino
(expresado ya sea mediante actos o palabras, que en esta sección prevalecen y
se identifican en general con el eje semántico de la verticalidad-descendente),
que reclama una respuesta humana, que, como consecuencia del libre albedrío,
puede ser positiva (mediante la obediencia y aceptación de la voluntad de Dios,
ejemplificada en estas estrofas por Aarón, Gedeón, los profetas y la prefiguración de María) o la negativa (los «falsos lesongeros» –4c– y los judíos incrédulos –15cd y 17d–, descriptos en la subsección referida a las figuras propiamente cristológicas).
En lo que concierne al entramado semántico-alegórico de la secuencia, se
ha examinado la recurrencia del esquema de contraposición de fuerzas antagónicas en lo que atañe a las relaciones entre lo divino-santo y lo humano. De
esta manera se verifica en esta instancia una pauta estructural de la obra, que
expresa el dinamismo de la historia de la salvación y la respuesta humana. Se
ha indicado la identificación de cada pasaje que contuviera esta carga semántica con el plano divino-santo correspondiente y su direccionalidad, a saber: la
horizontalidad-centrífuga de la misericordia y donación divinas, identificada
con el sema de la ‘apertura’18 y la verticalidad-descendente del poder divino,
representada en estos pasajes por los distintos signos y discursos proféticos e
18 La horizontalidad-centrífuga suele estar representada por María y sus cualidades, tal
como se ha estudiado en estas figuras, pero también muy presente en la conclusión del
poema. Sin embargo, cabe destacar que el hecho de la historia de la salvación que mayormente se identifica con este eje es la Pasión, acción axial de la misericordia y el amor de
Dios, relatado en las cuadernas 54-77.
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identificada con el sema de la ‘clausura’19. Ambas direccionalidades de fuerzas
encuentran su punto de contención en la figura de Cristo, como fruto paradójico de una madre virginal. En consecuencia, este esquema evidencia que la
fuerza ilocutiva de estas figuras se encuentra primariamente en explicar y alabar el rol instrumental e intercesor de María en la economía de la salvación.
Bibliografía citada
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En el relato posterior del Nuevo Testamento, se sumarán otros signos y los milagros:
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(124-127) y los siete dones del Espíritu Santo en Pentecostés (153-159).
19
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Fecha de recepción: 18 de abril de 2019.
Fecha de aceptación: 5 de septiembre de 2019.
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https://zenodo.org/records/7552033/files/105-113.pdf
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SUV TA'MINOTI TIZIMLARINI MASOFAVIY BOSHQARISH
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Zenodo (CERN European Organization for Nuclear Research)
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SUV TA'MINOTI TIZIMLARINI MASOFAVIY BOSHQARISH SUV TA'MINOTI TIZIMLARINI MASOFAVIY BOSHQARISH
Abror To'raqulov Safarovich
Toshkent kimyo-texnologiya instituti Shahrisabz filiali katta o’qituvchisi
Choriyev Abdirauf Mahmarejab oʻgʻli
Toshkent kimyo texnologiya instituti Shahrisabz filiali talabasi
asturaqulov@gmail.com
https://doi.org/10.5281/zenodo.7552033 Annotatsiya: Ushbu maqola Suv ta'minoti tizimlarini masofaviy boshqarish haqida bo’lib
mavzu yuzasidan tadqiqotchi olimlarning fikr va mulohazalari chuqur o’rganib chiqildi. Bugungi
global iqlim o’zgarishi sharoitida suv resurslaridan samarali foydalanish maqsadida suvtejamkor
texnologiyalardan foydalanishning xorijiy davlatlar tajribasi o’rganildi va bunda suvtejamkor
texnologiyalarning afzalliklari va qo’llanilishi bo’yicha takliflar ishlab chiqilgan. Kalit so’zlar: iqlim o’zgarishi, suv tanqisligi, suv limiti, suv xo’jaligi, raqamli
texnologiyalar, suvtejamkor texnologiyalar, tomchilatib sug’orish, suv xo’jaligiga bozor
tamoyillarini joriy qilish, sug’oriladigan yerlar. ДИСТАНЦИОННОЕ УПРАВЛЕНИЕ СИСТЕМАМИ ВОДОСНАБЖЕНИ ДИСТАНЦИОННОЕ УПРАВЛЕНИЕ СИСТЕМАМИ ВОДОСНАБЖЕНИЯ Аннотация:
Эта
статья
посвящена
удаленному
управлению
системами
водоснабжения, и были подробно изучены мнения и мнения ученых-исследователей по
этому вопросу. В целях эффективного использования водных ресурсов в условиях
современных глобальных изменений климата изучен опыт зарубежных стран по
использованию водосберегающих технологий, выработаны предложения о преимуществах
и применении водосберегающих технологий. Ключевые слова: изменение климата, дефицит воды, лимит воды, управление
водными ресурсами, цифровые технологии, водосберегающие технологии, капельное
орошение, внедрение рыночных принципов управления водными ресурсами, орошаемые
земли. REMOTE CONTROL OF WATER SUPPLY SYSTEMS Abstract: This article is about the remote management of water supply systems, and the
opinions and opinions of research scientists on the subject were studied in depth. In order to
effectively use water resources in the conditions of today's global climate change, the experience
of foreign countries in using water-saving technologies was studied, and proposals were developed
on the advantages and application of water-saving technologies. Keywords: climate change, water scarcity, water limit, water management, digital
technologies, water-saving technologies, drip irrigation, introduction of market principles in water
management, irrigated lands. Research Focus, Uzbekistan RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) | VOLUME 2 | ISSUE 1 | 2023
| Google Scholar | SJIF (4.597) | UIF (8.3) RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) | VOLUME 2 | ISSUE 1 | 2023
| Google Scholar | SJIF (4.597) | UIF (8.3) Respublikada 2020 — 2030 yillarda aholini va iqtisodiyotning barcha tarmoqlarini suv
bilan barqaror ta’minlash, sug’oriladigan yerlarning meliorativ holatini yaxshilash, suv xo’jaligiga
bozor tamoyillari va mexanizmlarini hamda raqamli texnologiyalarni keng joriy etish, suv
xo’jaligi ob’ektlarining ishonchli ishlashini ta’minlash hamda yer va suv resurslaridan foydalanish
samaradorligini oshirishga qaratilgan kontseptsiyani ishlab chiqilishi aynan suv xo’jaligi sohasida
olib borilayotgan islohotlar natijasidir. Ayniqsa bugungi suv tanqisligi sharoitida qishloq xo’jaligi ekinlarini yetishtirishda suv
tejovchi sug’orish texnologiyalarini joriy qilishni yanada kengaytirish va davlat tomonidan
rag’batlantirish, ushbu sohaga xorijiy investitsiyalar va grantlarni jalb qilish masalalarini amalga
oshirish dolzarb vazifalardan sanaladi. Ma’lumki, iqlim o’zgarishi O’zbekistonda suv taqchilligini yanada keskinlashtirishini,
2000, 2008, 2011, 2014 va 2018 yillardagi kabi qurg’oqchilikning davomiyligi va davriyligi
ko’payishiga olib kelishini hamda iqtisodiyotning suv resurslariga bo’lgan ehtiyojini qondirishda
jiddiy qiyinchiliklarni keltirib chiqarishi mumkinligini ko’rsatmoqda. Keyingi 15 yil ichida aholi
jon boshiga suv ta’minoti 3 048 kub metrdan 1 589 kub metrga qisqardi. Shu bilan birgalikda,
respublikada aholi soni yiliga o’rtacha 650 — 700 ming nafarga oshib, 2030 yilga borib 39 mln
nafarga yetishi, ularning sifatli suvga bo’lgan talabi 2,3 mlrd kub metrdan 2,7 — 3,0 mlrd kub
metrga (18 — 20 foiz) yetishi kutilmoqda. Shu nuqtai nazardan suvtejamkor texnologiyalardan foydalanishda xorijiy tajribalarning
ahamiyatli jihatlarini ayrim davlatlar misolida ko’rib chiqamiz va uni mamlakatimizning
sug’oriladigan yerlarida foydalanish bo’yicha taklif va tavsiyalar beramiz. TADQIQOT MATERIALLARI VA METODOLOGIYASI Tadqiqot jarayonida qiyosiy taqqoslash, mantiqiy va abstrakt
fikrlash usullaridan
foydalanildi. Research Focus, Uzbekistan KIRISH So’nggi yillarda yer va suv resurslaridan samarali foydalanish, suv resurslarini boshqarish
tizimini takomillashtirish, suv xo’jaligi ob’ektlarini modernizatsiya qilish va rivojlantirish
bo’yicha izchil islohotlar amalga oshirilmoqda. Shu bilan birga, global iqlim o’zgarishi, aholi
sonining va iqtisodiyot tarmoqlarining o’sishi, ularning suvga bo’lgan talabi yil sayin oshib borishi
tufayli suv resurslarining taqchilligi yildan-yilga kuchayib bormoqda. Foydalanilgan o’rtacha yillik
suv miqdori 51 — 53 mlrd kub metrni, jumladan, 97,2 foizi daryo va soylardan,1,9 foizi kollektor
tarmoqlaridan, 0,9 foizi esa yer ostidan foydalanib, ajratilgan suv olish limitiga nisbatan 20 foizga
qisqargan. Research Focus, Uzbekistan 105 refocus.uz Research Focus, Uzbekistan MQTT protokoli haqida umumiy ma’lumot Message Queuing Telemetry Transport (MQTT) protokoli ko'p yillar davomida mavjud
bo'lib kelgan, ammo hozirda bu IoTning portlovchi o'sishi tufayli juda dolzarbdir: iste'molchilar
ham, ishlab chiqaruvchilar ham tarqatilgan tarmoqlarni va chekka hisoblashlarni qabul
qilmoqdalar va doimiy ma'lumotlar uzatuvchi qurilmalar kundalik hayotning bir qismiga aylanish. Bu shuni anglatadiki, engil, ochiq va qulay protokollar vaqt o'tishi bilan yanada muhimroq
bo'ladi. Ushbu maqola MQTT-ga kontseptual sho'ng'in beradi: u qanday ishlaydi, hozir qanday
ishlatiladi va kelajakda qanday foydalaniladi. MQTT asosida qurilgan aloqa tizimi nashriyot serveridan, broker serveridan va bir yoki bir
nechta mijozlardan iborat. Nashriyot xabar olgan abonentlarning soni yoki joylashuvi bo'yicha
hech qanday o'zgartirish kiritishni talab qilmaydi. Bundan tashqari, obunachilar ma'lum bir noshir
uchun sozlanishi shart emas. Tizimda xabarlarni tarqatadigan bir nechta broker bo'lishi mumkin. Research Focus, Uzbekistan 106 refocus.uz refocus.uz RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) | VOLUME 2 | ISSUE 1 | 2023
| Google Scholar | SJIF (4.597) | UIF (8.3) p (
)
|
g
|
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) |
(
) MQTT aloqa kanallari iyerarxiyasini yaratish yo'lini taqdim etadi - bu barglar bilan bir xil
filial. Nashriyot xaridorlarga tarqatish uchun har doim yangi ma'lumotlarga ega bo'lganda, xabar
etkazib berishni nazorat qilish yozuvlari bilan birga keladi. Yuqori darajadagi mijozlar har bir
xabarni, pastki darajadagi mijozlar esa ierarxiyaning pastki qismida joylashgan bitta yoki ikkita
asosiy kanalga tegishli xabarlarni qabul qilishlari mumkin. Bu ikki baytdan 256 megabaytgacha
bo'lgan hajmdagi ma'lumot almashinuvini osonlashtiradi. MQTT brokeri orqali ulanish uchun mijozni qanday sozlash mumkinligiga misol: MQTT brokeri orqali ulanish uchun mijozni qanday sozlash mumkinligiga misol:
var options = {
keepalive: 60,
username: 'FIRST_HALF_OF_API_KEY',
password: 'SECOND_HALF_OF_API_KEY',
port: 8883 };
var client = mqtt.connect('mqtts:mqtt.ably.io', options);
MQTT b
k i t
id
h
til
ki
li
h
d
'l
tl
ikkili MQTT brokeri orqali ulanish uchun mijozni qanday sozlash mumkinligiga misol:
var options = { username: FIRST_HALF_OF_API_KEY ,
password: 'SECOND_HALF_OF_API_KEY',
port: 8883 }; var client = mqtt.connect('mqtts:mqtt.ably.io', options); MQTT brokeri tomonidan nashr etilgan yoki olingan har qanday ma'lumotlar ikkilik
kodlangan bo'ladi, chunki MQTT ikkilik protokol hisoblanadi. Bu shuni anglatadiki, asl tarkibni
olish uchun siz xabarni sharhlashingiz kerak. Ably va JavaScript bilan shunday ko'rinadi: MQTT brokeri tomonidan nashr etilgan yoki olingan har qanday ma'lumotlar ikkilik
kodlangan bo'ladi, chunki MQTT ikkilik protokol hisoblanadi. Bu shuni anglatadiki, asl tarkibni
olish uchun siz xabarni sharhlashingiz kerak. refocus.uz Research Focus, Uzbekistan client.subscribe("[mqtt]tokenevents", { /* Create a new token called 'NEW_TOKEN' */
client.end();
options.username = NEW_TOKEN;
client = mqtt.connect('mqtts:mqtt.ably.io', options);
});
MQTT funktsionalligi: chuqurroq sho'ng'in
IBM ma'lumotlariga ko'ra MQTT quyidagi xususiyatlarga ega:
Xabar tarkibi uchun neytral
Bir-biridan tarqatilgan aloqa va uzilgan dasturlar uchun ideal
Mijozning g'ayritabiiy ravishda uzilishi haqida tomonlarga xabar berish uchun LWT
(Oxirgi vasiyat va vasiyat) funktsiyasi bilan jihozlangan
Asosiy aloqa vazifalari uchun TCP / IP-ga tayanadi
Xabarlarni "maksimal bir marta", "minimal bir marta" va "to'liq bir marta" naqshlari
bo'yicha etkazib berish uchun mo'ljallangan IBM ma'lumotlariga ko'ra MQTT quyidagi xususiyatlarga ega: MQTT tizimining ishtirokchisi noshir, iste'molchi yoki har ikkalasi vazifasini bajarishi
mumkin. MQTT tizimining ishtirokchisi noshir, iste'molchi yoki har ikkalasi vazifasini bajarishi
mumkin. MQTT-ning ajralib turadigan xususiyatlaridan biri bu kanallarni noyob tushunishi:
ularning har biri faylga yo'l sifatida ko'rib chiqiladi, masalan: MQTT-ning ajralib turadigan xususiyatlaridan biri bu kanallarni noyob tushunishi:
ularning har biri faylga yo'l sifatida ko'rib chiqiladi, masalan: Kanallar har bir mijoz o'zi uchun mo'ljallangan xabarlarni qabul qilishini ta'minlaydi. Quvurlarni fayl yo'llari sifatida ko'rib chiqish orqali MQTT har xil foydali aloqa funktsiyalarini
bajaradi, shu jumladan xabarlarni qayerda - qaysi darajada yoki qaysi filialda bo'lishiga qarab
mijozlar fayl yo'liga obuna bo'lishlari asosida filtrlash. MQTT protokoli haqida umumiy ma’lumot Ably va JavaScript bilan shunday ko'rinadi: g
y
p
y
var ably = new Ably.Realtime('REPLACE_WITH_YOUR_API_KEY');
var decoder = new TextDecoder();
var channel = ably.channels.get('input');
channel.subscribe(function(message) {
var command = decoder.decode(message.data); }); var ably = new Ably.Realtime('REPLACE_WITH_YOUR_API_KEY');
var decoder = new TextDecoder(); var ably = new Ably.Realtime('REPLACE_WITH_YOUR_API_KEY');
var decoder = new TextDecoder();
var channel = ably.channels.get('input');
h
l
b
ib (f
i
(
) { var channel = ably.channels.get('input'); channel.subscribe(function(message) { var command = decoder.decode(message.data); }); MQTT brokerlari ba'zida amaldagi obunachilari bo'lmagan kanallar bilan bog'liq
xabarlarni to'plashlari mumkin. Bunday holda, xabarlar boshqaruv xabaridagi ko'rsatmalarga
qarab yo'q qilinadi yoki saqlanadi. Bu yangi abonentlarga keyingi jo'natishni kutish o'rniga eng MQTT brokerlari ba'zida amaldagi obunachilari bo'lmagan kanallar bilan bog'liq
xabarlarni to'plashlari mumkin. Bunday holda, xabarlar boshqaruv xabaridagi ko'rsatmalarga
qarab yo'q qilinadi yoki saqlanadi. Bu yangi abonentlarga keyingi jo'natishni kutish o'rniga eng 107 refocus.uz refocus.uz RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) | VOLUME 2 | ISSUE 1 | 2023
| Google Scholar | SJIF (4.597) | UIF (8.3) so'nggi qayd qilingan ma'lumotlar nuqtasi kerak bo'lishi mumkin bo'lgan hollarda foydalidir. ggi qayd qilingan ma'lumotlar nuqtasi kerak bo'lishi mumkin bo'lgan hollarda foydalidir. Ta'kidlash joizki, MQTT xavfsizlik ma'lumotlarini aniq matnda uzatadi, aks holda
autentifikatsiya yoki xavfsizlik xususiyatlari qo'llab-quvvatlanmaydi. Bu erda SSL ramkasi
o'ynaladi, bu uzatilayotgan ma'lumotni ushlanib qolishdan yoki boshqa yo'l bilan buzilishdan
himoya qilishga yordam beradi. Bundan tashqari, API kalitingizni haqiqiy MQTT mijoziga ko'rsatishni istamasangiz, Ably
token autentifikatsiyasi MQTT-da ishlatilishi mumkin (agar SSLsiz MQTT bo'lsa, API kalitlarini
aniq matnda uzatishni oldini olish uchun tokenlar talab qilinadi ). Tokenlar orqali autentifikatsiya
qilishga misol: var options = { keepalive: 60, username: INSERT_TOKEN_HERE,
password: '',
port: 8883 };
var
client
=
m username: INSERT_TOKEN_HERE, username: INSERT_TOKEN_HERE,
password: '',
port: 8883 };
var
client
=
mqtt.connect('mqtts:mqtt.ably.io',
options);
client.subscribe("[mqtt]tokenevents", { client.subscribe("[mqtt]tokenevents", { RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) | VOLUME 2 | ISSUE 1 | 2023
| Google Scholar | SJIF (4.597) | UIF (8.3) Turkiya iqlimi qishloq xo’jaligi uchun juda ko’p qulayliklar yaratadi va bu barcha turdagi
qishloq xo’jaligi mahsulotlarini yetishtirish imkonini beradi. Ammo qishloq xo’jaligi tarmog’i
yaxshi rivojlanmagan, masalan, katta imkoniyatlar mavjud bo’lgan sharoitda asosiy qishloq
ho’jaligi ekini bo’lgan g’allaning o’rtacha hosildorligi 21 tsentnerni tashkil qiladi va aholi jon
boshiga yetishtiriladigan go’sht miqdori o’rtacha 18 kg tashkil qiladi. Suv xo’jaligi bilan bog’liq barcha ishlarni rejalashtirish, loyihalash, qurish, suv
toshqinlariga qarshi kurash, qishloq xo’jaligi ekinlari maydonlarini suv bilan ta’minlash, suv
resurslarini shahar va qishloqlarga yetkazib berish ishlarini Davlat suv xo’jaligi boshqarmasi
(DSI) amalga oshiradi. Shu bilan bir qatorda, suv bilan bog’liq bajariladigan va bajarilishi lozim
bo’lgan ishlar bilan shaxsan Bosh Vazir shug’ullanadi va nazorat olib boradi. Turkiyada qishloq xo’jaligi ekinlarini sug’orish maqsadlarida ishlatiladigan suvga
to’lovlar bo’yicha mahalliy sharoit hisobga olingan mexanizm ishlab chiqilgan. Sug’oriladigan
yerlarning 55,8 foizi eski sug’oriladigan va 44,2 foizi yangidan o’zlashtirilgan yerlar bo’lib, eskidan sug’oriladigan yerni sug’orish uchun ishlatiladigan suvga xaq olinmaydi. Bunda
ekin maydonlarini sug’orish va suvni taqsimlash, suvdan foydalanish inshootlari qadimdan
mavjud bo’lganligi e’tiborga olingan. Hozirgi kunda Turkiyada 152 ta suv ombori ishlab turibdi,
yana yangidan 50 dan ziyod suv omborlari qurilmoqda. Ahamiyatli jihati shundaki, davlat
tomonidan suv xo’jaligi tizimi faoliyati uchun sarflanayotgan umumiy mablag’larning 40
foizidan ortig’i gidroenergetika resurslarini sotish hisobidan qoplanadi. Mamlakatda
sug’oriladigan yerlaning ahamiyatli jihatlari juda katta bo’lib, bunday yerlarni aholiga uy-joy
qurish yoki boshqa maqsadlar uchun taqsimlashtirish ishlari qattiq nazoratga olingan. Xitoy davlatining haydaladigan yer maydoni 100 mln. gektar bo’lib, shundan 50 mln. gektari sug’oriladigan yerlar hisoblanadi. Yillik yog’ingarchilik miqdori o’rtacha 1200 mm.ni
tashkil qiladi yoki har gektar yerga 12 ming kubometr suv zahirasi to’g’ri keladi. Bu yerda
sug’orish ishlari uchun bir yilda o’rtacha 400 mlrd. kubometr suv ishlatilib, o’rtacha bir gektar
maydonga 7,1 ming kubometr suv zaxirasi to’g’ri keladi. Xitoydagi yirik suv xo’jaligi tizimlaridan
biri Xuanxe daryosi asosida faoliyat yurituvchi majmuadir. Majmuaning uzunligi 252 kilometr
bo’lib, u 460 ta suv inshootlari, 13 ta dyukerni o’z ichga oladi. Bu yerda suv daryodan 40 metr
balandlikka ko’tarib beriladi. Bugungi kunda Xitoy suv xo’jaligi vazirligi suvdan foydalanish
borasida katta huquqlarga ega bo’lib, mamlakatda suv xo’jaligi bo’yicha alohida militsiya tizimi
ham tashkil qilingan va bu suvdan foydalanish jarayonini nazorat qiladi. Amerika Qo’shma
Shtatlarida 1960 yillarning boshida Richard Xapin tomonidan “Shudringli shlang” nomi (boshqa
nomi “spagetti quvuri”) bilan tomizgichli lenta ishlab chiqildi va uning birinchi namunasi 1964
yilda amaliyotga joriy qilindi. Research Focus, Uzbekistan MUHOKAMA VA NATIJALAR Qishloq xo’jaligida suvdan foydalanish borasida Turkiya tajribasi alohida ahamiyatga
molikdir. Xususan, Turkiyaning o’rtacha yillik suv resurslari 180-190 milliard kubometrni, yer
osti suvlari esa 10-15 milliard kubometrni, jami suv resurslari esa 200 milliard kubometrni tashkil
qilsa-da, uning faqatgina 30-35 mlrd. m3 (15 foizi) ishlatiladi. Shundan oqova suv resurslari 25-
26 mlrd. m3 va yer osti suvlari resurslar 5-6 mlrd. m3. 108 refocus.uz RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) Bunday shlanglar asosan daraxtlarni va issiqxonalarda
yetishtiriladigan gullarni sug’orish uchun keng joriy qilindi. Tomchilatib sug’orish tizimlaridan foydalanish 1980 yildan keyin ayniqsa kuchaydi va
2000 yilga kelib dunyo miqyosida tomchilatib sug’orish tizimlari joriy qilingan ekin maydonlari
3,2 mln. gektardan ortib ketdi. AQShning Xavay orollarida qiyalik maydonlarda shakarqamish
yetishtirishda egatdan sug’orish usulini qo’llash mumkin bo’lmaganligi bois fermerlar
yomg’irlatib sug’orishdan foydalanishgan va katta qiyinchiliklarga duch kelishgan. Tomchilatib
sug’orish yaxshi samara berishi aniqlangandan so’ng esa Xavaydagi 11 shakarqamish plantatsiyasi
1986 yilda to’laligicha tomchilatib sug’orishga o’tkazilgan. Tomchilatib sug’orish tizimlarini qo’llashda Isroil, Kipr, AQSh, Italiya, Avstraliya va
Iordaniya kabi jahon mamlakatlarida juda katta yutuqlarga erishildi. AQSh, Avstraliya, Isroil va boshqa bir qator mamlakatlarda yer ostidan tomchilatib Research Focus, Uzbekistan 109 refocus.uz refocus.uz RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) | VOLUME 2 | ISSUE 1 | 2023
| Google Scholar | SJIF (4.597) | UIF (8.3) sug’orish tizimlari ham keng tarqaldi. Ushbu tizimlar suvni o’simlikning ildiz qatlami ostidan
yetkazib berishga moslashtirilganligi bilan boshqa tomchilatib sug’orish tizimlaridan keskin farq
qiladi. Yevropa mamlakatlarida suv resurslaridan foydalanish va ularni samarali boshqarish
masalasi eng dolzarb mavzular qatoriga kiradi. Masalan, Ispaniyada keyingi yillarda qurg’oqchilik
sababli kelib chiqayotgan qator muammolar natijasida avvalroq vujudga kelgan og’ir ekologik
vaziyatni yaxshilash dasturlari, daryolarni bir-biriga oqizish tadbirlari amalga oshirilgan bo’lsa,
yaqin yillardagi yog’ingarchiliklar va suv toshqinlari tufayli, uzoq kelajakka mo’ljallangan kuchli
davlat tadbirlari ustida ish olib borilmoqda. sug’orish tizimlari ham keng tarqaldi. Ushbu tizimlar suvni o’simlikning ildiz qatlami ostidan
yetkazib berishga moslashtirilganligi bilan boshqa tomchilatib sug’orish tizimlaridan keskin farq
qiladi. Yevropa mamlakatlarida suv resurslaridan foydalanish va ularni samarali boshqarish
masalasi eng dolzarb mavzular qatoriga kiradi. Masalan, Ispaniyada keyingi yillarda qurg’oqchilik
sababli kelib chiqayotgan qator muammolar natijasida avvalroq vujudga kelgan og’ir ekologik
vaziyatni yaxshilash dasturlari, daryolarni bir-biriga oqizish tadbirlari amalga oshirilgan bo’lsa,
yaqin yillardagi yog’ingarchiliklar va suv toshqinlari tufayli, uzoq kelajakka mo’ljallangan kuchli
davlat tadbirlari ustida ish olib borilmoqda. Kuzatishlar shuni ko’rsatadiki, barcha davlatlarda XXI asr boshidan boshlab insoniyat
uchun global muammoga aylanib borayotgan suv resurslari taqchilligi masalasini hal qilishga,
ulardan oqilona foydalanishga e’tibor berib, o’z iqtisodiy ko’rsatkichlari, ichki va tashqi
imkoniyatlaridan kelib chiqqan holda dasturlar tayyorlamoqdalar va dasturlarni ma’lum darajada
amalga oshirmoqdalar. Suvdan foydalanish jarayoni tahlil qilinganda yana bir masalaga e’tibor qaratish lozim,
ya’ni dunyodagi davlatlardan birortasining qishloq xo’jaligida O’zbekiston kabi deyarli 100 foiz
sug’oriladigan yer maydonlaridan foydalanilmaydi. Markaziy Osiyo hududida, xususan,
O’zbekiston hududida yozda o’simliklarning vegetatsiya davrida yog’ingarchilik umuman
kuzatilmasligi tufayli tuproq namini ushlab turish faqatgina sug’orish tadbirlari orqali amalga
oshiriladi. Research Focus, Uzbekistan RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) Bu holat nafaqat mas’uliyatni, shuningdek katta mablag’ni sarflab turishni taqozo etadi. O’zbekiston sug’oriladigan yer maydonlari, bu maydonlarga ishlov berish va yuqori hosil
yetishtirish jarayonlarida suv xo’jaligi tizimining ahamiyati kattaligi bo’yicha ham, bu sohaga
davlat tomonidan ajaratilayotgan mablag’ miqdoriga ko’ra ham boshqa davlatlardan keskin farq
qiladi. Ma’lumki, rivojlangan davlatlarda sug’orish texnologiyasini takomillashtirishga katta
ahamiyat beriladi. Sug’orish texnologiyasining progressiv usullari dastlabki kapital quyilmalarni
ko’proq talab qilsa ham, suv sarfini va mehnat sarfini tejash imkoniyatini beradi. Bu, ayniqsa, ish
kuchi qimmat bo’lgan davlatlarda juda muhim ahamiyat kasb etadi. Ma’lumki, O’zbekiston sharoitida tomchilatib sug’orish tizimlari asosan 1975 yildan
boshlab tajriba tariqasida bog’ va uzumzorlarda tadbiq qilina boshlangan. Bu davrda, ya’ni 1975 yilda esa SANIIRI institutining Jizzax viloyati Zomin tumanidagi
tajriba xo’jaligida avval 10 ga, keyinchalik 200 ga maydondagi uzumzorni, 1977 yilda Xorazm
viloyatining Xiva tumanida 1,5 ga maydondagi bog’ni, Shreder nomidagi bog’dorchilik va
uzumchilik ilmiy tadqiqot institutining 2,0 ga maydondagi bog’ini sug’orish uchun mahalliy
sharoitlarda yaratilgan tomchilatib sug’orish tizimlari joriy qilingan. Tomchilatib sug’orish
tizimlarini qo’llash 1990 yillar boshida ancha kengaytirildi va ularning maydoni 1993 yilga kelib
1134 gektarga yetkazildi. Shu jumladan, 1991-1992 yillarda Isroil texnologiyasi asosida Andijon
viloyatining Qo’rg’ontepa tumanidagi “Savay” xo’jaligida 1 ming ga paxta maydonida 6,6 mln. AQSh dollari qiymatiga ega bo’lgan tomchilatib sug’orish tizimi joriy qilish ishlari olib borildi va
uning 500 gektarli qismi ishga tushirildi. Xuddi shu yillarda tomchilatib sug’orish tizimlarini paxta yetishtirishda qo’llash
mumkinligi o’rganildi. SANIIRIda olib borilgan tadqiqotlar natijalari paxta yetishtirishda
tomchilatib sug’orishni qo’llash egatlab sug’orishga nisbatan suvni 1,5 – 3,0 martagacha kamaytirish, paxtadan gektariga 35-43 tsentner miqdorida hosil olish
mumkinligini tasdiqladi. 1990 yillarning ikkinchi yarmida O’zbekistonda yana 600 ga maydonda
tomchilatib sug’orish tizimlari joriy qilindi. Shu jumladan 1999- 2001 yillarda Toshkent, Jizzax 110 refocus.uz refocus.uz RESEARCH FOCUS
| VOLUME 2 | ISSUE 1 | 2023
ISSN: 2181-3833 ResearchBip (14)
| Google Scholar | SJIF (4.597) | UIF (8.3) RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) | VOLUME 2 | ISSUE 1 | 2023
| Google Scholar | SJIF (4.597) | UIF (8.3) va Sirdaryo viloyatlarida uchta 100 gektarli maydonda Isroil davlati Netafim firmasining har biri
2,1 mln. AQSh dollari turadigan tomchilatib sug’orish tizimlari tadbiq qilindi. Qurilgan ushbu
sug’orish tizimlari turli sabablarga ko’ra ko’ngildagiday faoliyat yuritishmadi. O’zbekistonda 1975 - 2000 yillar oralig’ida qurilgan tomchilatib sug’orish tiziml O’zbekistonda 1975 - 2000 yillar oralig’ida qurilgan tomchilatib sug’orish tizimlaridan biri
- Qashqadaryo viloyati «Varganza» xo’jaligidagi anorzorni tomchilatib sug’orish tizimi (1990
yilda qurilgan) hozirgi kunda ham faoliyat yuritmoqda. Research Focus, Uzbekistan RESEARCH FOCUS
ISSN: 2181-3833 ResearchBip (14) O’zbekiston Respublikasi Prezidentining
2019 yil 25 oktyabrdagi - Qashqadaryo viloyati «Varganza» xo’jaligidagi anorzorni tomchilatib sug’orish tizimi (1990
yilda qurilgan) hozirgi kunda ham faoliyat yuritmoqda. O’zbekiston Respublikasi Prezidentining
2019 yil 25 oktyabrdagi «Qishloq xo’jaligida suv tejovchi texnologiyalarni joriy etishni rag’batlantirish
mexanizmlarini kengaytirish chora-tadbirlari to’g’risida»gi PQ-4499-sonli qarorida suvni
tejaydigan texnologiyalarni joriy etish uchun davlat byudjetidan 300 mlrd so’m subsidiya
ajratilishi belgilandi va bunda tomchilatib sug’orish tizimlari uchun - 8 mln so’m, yomg’irlatib
sug’orish tizimlari uchun - 4 mln so’m, diskretli sug’orish uchun - 1 mln so’m mablag’ ajratilishi
belgilandi. 2013-2019 yillarda respublikada jami 76,2 ming gektar qishloq xo’jaligi ekin maydonlarida
shundan, 52,5 ming gektar bog’ va uzumzorlarda, 11,9 ming gektar sabzavot va poliz mahsulotlari
hamda 11,7 ming gektar paxta maydonlarda tomchilatib sug’orish texnologiyasi joriy etildi. 1-rasm. Suv tejovchi texnologiyalarni qo’llash ko’rsatkichlari 1-rasm. Suv tejovchi texnologiyalarni qo’llash ko’rsatkichlari Ushbu rasmdan ham ko’rinib turibdiki, mamlakatimiz qishloq xo’jaligida suvtejamkor
texnologiyalarni qo’llash samaradorligi va 2025 yilgacha bo’lgan prognoz ko’rsatkichlari
keltirilgan bo’lib, bugungi global iqlim o’zgarishi va suv tanqisligi sharoitida sug’orishning
suvtejamkor texnologiyalarni qo’llash davr talabi ekanligini ko’rsatmoqda. Ushbu rasmdan ham ko’rinib turibdiki, mamlakatimiz qishloq xo’jaligida suvtejamkor
texnologiyalarni qo’llash samaradorligi va 2025 yilgacha bo’lgan prognoz ko’rsatkichlari
keltirilgan bo’lib, bugungi global iqlim o’zgarishi va suv tanqisligi sharoitida sug’orishning
suvtejamkor texnologiyalarni qo’llash davr talabi ekanligini ko’rsatmoqda. XULOSA Xulosa qiladigan bo’lsak, xorijiy davlatlarda sug’orish texnologiyasini takomillashtirishga
katta ahamiyat berilgan. Dunyodagi ko’pgina mamlakatlarning har bir melioratsiya va suv
xo’jaligi bo’yicha o’z tarixiy an’analariga, suv resurslariga bo’lgan ehtiyojiga, iqtisodiyotning
rivojlanish yo’liga, sug’orish tarixiga egadir va ular bir - biridan farq qiladi. Suvdan foydalanish
yo’nalishlari ularda, asosan, davlatning taraqqiyot darajasiga qarab belgilangan. Mamlakatimizda, suvni tejovchi texnologiyalarni keng joriy qilish, bunda davlat
tomonidan yaratilayotgan qulayliklardan foydalanish qishloq xo’jaligi ishlab chiqarishini yanada 111 refocus.uz refocus.uz | VOLUME 2 | ISSUE 1 | 2023
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ISSN: 2181-3833 ResearchBip (14) rivojlantirish maqsadida quyidagi takliflarni bermoqchimiz: suv resurslarini boshqarish tizimini takomillashtirish, suvdan foydalanish va suv
iste’moli hisobini yuritishda «Smart Water» («Aqlli suv») va shu kabi raqamli texnologiyalarni
joriy qilish; suv resurslarini boshqarish tizimini takomillashtirish, suvdan foydalanish va suv
iste’moli hisobini yuritishda «Smart Water» («Aqlli suv») va shu kabi raqamli texnologiyalarni
joriy qilish; qishloq xo’jaligi ekinlarini yetishtirishda suv tejovchi sug’orish texnologiyalarini joriy
qilishni yanada kengaytirish va davlat tomonidan rag’batlantirib borish, ushbu sohaga xorijiy
investitsiyalar va grantlarni jalb qilish; qishloq xo’jaligi ekinlarini yetishtirishda suv tejovchi sug’orish texnologiyalarini joriy
qilishni yanada kengaytirish va davlat tomonidan rag’batlantirib borish, ushbu sohaga xorijiy
investitsiyalar va grantlarni jalb qilish; sug’oriladigan yerlarning meliorativ holatini yaxshilash va barqarorligini ta’minlash,
yerlarning unumdorligini oshirishga ko’maklashish, tuproqning sho’rlanish darajasini pasaytirish
va oldini olish bo’yicha samarali texnologiyalarni qo’llash; suv xo’jaligida bozor iqtisodiyoti tamoyillarini, jumladan, suvni yetkazish
xarajatlarining bir qismini bosqichma-bosqich suv iste’molchilari tomonidan qoplash tizimini
joriy qilish, tushgan mablag’larni suv xo’jaligi ob’ektlarini o’z vaqtida sifatli ta’mirlash-tiklash,
raqamli texnologiyalarni joriy qilish hamda samarali boshqarishga yo’naltirish; suv xo’jaligida davlat-xususiy sheriklik va autsorsingni joriy etish, alohida suv
xo’jaligi ob’ektlarini fermer, klaster va boshqa tashkilotlarga foydalanish uchun berish hamda
tejalgan mablag’larni suv xo’jaligi ob’ektlarini modernizatsiya qilish va xodimlar mehnatiga haq
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UTILIZATION OF SOCIAL MEDIA AS A QUARTER LIFE CRISIS EDUCATION MEDIA
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Inovasi Lokal Inovasi Lokal
Pemberdayaan Masyarakat dalam Pembangunan Berkelanjutan
Tarqabin
Nusantara
Inovasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023
E-ISSN : 3024-9716 Pemberdayaan Masyarakat dalam Pembangunan Berkelanjutan Inovasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023 Dandy Arie Pramudya1 , Fathimah Az-Zahra Hayuningrat2 , Khansa Salsabila Putri
Ariyanto3 , Lubna Salsabila Esrarf 4 , Nisrina Alifah Naia5 , Sheilla Rahmayanti6 ,
Yasmin Nazilah7 1 Universitas Negeri Malang, Malang, Jawa Timur 1 Universitas Negeri Malang, Malang, Jawa Timur Correspondence: dandy.arie.2206126@students.um.ac.id Correspondence: dandy.arie.2206126@students.um.ac.id Responsible Editor: Ronal Surya Aditya INTRODUCTION Humans are transitional creatures where each developmental process has its own challenges
in life. When in late adolescence, humans will experience a transition to the adult phase or more
precisely early adulthood, namely the age of 20-30 years. Early adulthood is called the Quarter Life
Crisis (Muttaqien & Hidayati, 2020). q
y
According to Robbins and Wilner (2001) a quarter life crisis is an individual response that
shifts towards the reality of life in which there is instability, continuous change, there are various q
y
According to Robbins and Wilner (2001) a quarter life crisis is an individual response that
shifts towards the reality of life in which there is instability, continuous change, there are various
choices and the emergence of panic because they feel helpless. This kind of life change is q
y
According to Robbins and Wilner (2001) a quarter life crisis is an individual response that
shifts towards the reality of life in which there is instability, continuous change, there are various
choices and the emergence of panic because they feel helpless. This kind of life change is
accompanied by the emergence of various kinds of emotional reactions such as anxiety, panic,
stress, and confusion about goals, as well as doubts about one's own abilities and fear of failure shifts towards the reality of life in which there is instability, continuous change, there are various
choices and the emergence of panic because they feel helpless. This kind of life change is
accompanied by the emergence of various kinds of emotional reactions such as anxiety, panic,
stress, and confusion about goals, as well as doubts about one's own abilities and fear of failure
(Salsabila, 2021). Quarter Life Crisis is often experienced by students because they are in early adulthood where
they begin to dare to live their own lives. The problem that occurs is about questions about life after
graduating from college, where to go after college, will you get an established job (Muttaqien &
Hidayati, 2020). The advancement of technology, namely the existence of social media, has the advantage of
connecting various kinds of people. We can see their lives without meeting them in person. This
can have both good and bad effects, by seeing these contents we can feel the social gap. Inovasi Lokal
Pemberdayaan Masyarakat dalam Pembangunan Berkelanjutan
Tarqabin
Nusantara
asi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023
E-ISSN : 3024-9716 Inovasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023 Inovasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023
E-ISSN : 302 This is an open access article under Creative Commons
Attribution-ShareAlike 4.0 International License ABSTRACT Introduction : Quarter life crisis is an individual's response to moving towards the reality of life in
which there is instability, continuous change, various choices and the emergence of panic due to
feeling helpless. Life changes like this are accompanied by the emergence of various kinds of
emotional reactions such as anxiety, panic, stress, and confusion about goals, as well as doubts about
one's own abilities and fear of failure. Methods: in utilizing social media, using qualitative methods,
which include collecting data by interviews and delivering material. Purpose: to take an approach
involving visual narratives, interviews, and practical solutions in providing a comprehensive view of
the quarter life crisis and providing positive value in using social media and having a good impact on
oneself. Results: The author's video has its own interest among Instagram users, as evidenced by the
author's video views reaching 3,753 and video likes reaching 89 likes. This shows that Instagram users
gain knowledge about the quarter life crisis. Conclusion: Currently there are still students who
experience changes in emotional reactions such as anxiety, panic, stress, and confusion and doubt
about their own abilities. Video about the quarter life crisis by conducting interviews with students to
get various perspectives on the quarter life crisis and strategies for dealing with it. The video content
will be disseminated via social media such as Instagram. KEYWORDS
Instagram, Social media, Students, Quarter Life Crisis
Received: 11 October 2023
Revised: 10 November 2023
Accepted: 31 Desember 2023
How to cite: Pramudya, Dandy Arie et al. (2023). Utilization of Social Media as a Quarter Life Crisis
Education Media. Inovasi Lokal, 1(2): 135-140. KEYWORDS
Instagram, Social media, Students, Quarter Life Crisis 2023
2023
er 2023
How to cite: Pramudya, Dandy Arie et al. (2023). Utilization of Social Media as a Quarter Life Crisis
Education Media. Inovasi Lokal, 1(2): 135-140. Received: 11 October 2023
Revised: 10 November 2023
Accepted: 31 Desember 2023 135 This is an open access article under Creative Commons
Attribution-ShareAlike 4.0 International License Inovasi Lokal Pemberdayaan Masyarakat dalam Pembangunan Berkelanjutan
Nusa Inovasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023 understand, and overcome the crisis in the quarter-life phase of college students. Communication
through comments and direct messages provides a space for direct interaction and exchange of
experiences between students who face similar challenges. The @phoffedu Instagram account is
not only a place to understand the quarter-life crisis, but also an educational resource to manage the
quarter-life crisis well. understand, and overcome the crisis in the quarter-life phase of college students. Communication
through comments and direct messages provides a space for direct interaction and exchange of
experiences between students who face similar challenges. The @phoffedu Instagram account is
not only a place to understand the quarter-life crisis, but also an educational resource to manage the
quarter-life crisis well. The author has created content about the quarter-life crisis that has been presented on the
@phoffedu Instagram account. The content is in the form of video reels with a duration of six minutes
which begins with a scene of two female students who are experiencing a quarter-life crisis. Then followed
by interviews with several students from the Faculty of Sports Science, State University of Malang. The
interviews aimed to get diverse perspectives on the experience of quarter life crisis among students,
highlighting the challenges and strategies they chose in dealing with it. By involving university students
the content became more contextualized and relevant for audiences who may experience similar
situations. After conducting the interviews, at the end of the video the authors present various measures
that can be taken to overcome the quarter-life crisis, providing inspiration and practical guidance for
those facing this crucial phase in their lives. With an approach that involves visual narratives, interviews,
and practical solutions, this content aims to provide a comprehensive view of the quarter life crisis and
add educational value to Instagram users. INTRODUCTION One
example of a social media platform that is often used is Instagram, according to (Akhmad & Prili,
2018) The negative impact of Instagram for teenagers is a crisis of confidence, competition for
luxury life and unwillingness to accept reality. In this case, today's teenagers always follow the
trends that are taking place in the world and among them, because they do not want to be considered
outdated by their friends and are considered popular if they follow the times (Permatasari. A.,
Marsa. Ammar. Mohammad., 2022) Generation Z grew up with the social web, they are digital-centric and technology is their
identity (Singh & Dangmei, 2016). Generation Z can also be known as digital natives, they are used
to using digital media for various purposes, for example, "developing and maintaining connections,
building self-image, expressing thoughts and emotions, and seeking entertainment" (Nuzulita &
Subriadi, 2020, p. 1). Siska Kusuma Ningsih (2016) said that generation Z is a generation that has
a strong relationship with social media, and this has an impact on the way they think and also the
way they see life. It is also about how they live their lifestyles (Permatasari. A., Marsa. Ammar. Mohammad., 2022). Instagram has evolved into an educational platform through its prominent features of visual
presentation focusing on images and videos. Instagram users can utilize stories, reels, and
Instagram live to share knowledge, skills, and information in an engaging way, and can deliver
educational content in a more dynamic and structured format. The ability to add captions and long
descriptions to each post provides further context and depth to the material being shared. The
interaction that occurs through comments, direct messages and other interactive features allows for
discussion and exchange of ideas between content creators and audiences (Fitriani, 2021). Thus,
Instagram is not just a social media or entertainment platform, but also a dynamic and accessible
source of learning. Instagram @phoffeedu exists as an educational platform that discusses and provides guidance
related to the quarter life crisis, especially for college students. Through image and video
visualizations, the @phoffeedu Instagram account presents informative content that details the
various challenges and changes that are often experienced by students in the quarter-life crisis
phase. By utilizing Instagram features, the @phoffeedu account invites Instagram users to 136 Inovasi Lokal Pemberdayaan Masyarakat dalam Pembangunan Berkelanjutan
Nusa Inovasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023 population aged 18-24 years has the second highest percentage of social media use (15.54% for
women and 16.6% for men). This indicates that teenagers' enthusiasm for using social media is high
(Ayu Khoirotul Umaroh et al., 2023). The use of social media can be used as a place for education, as well as a campaign for the
public. Nowadays, the use of Instagram is quite common among young people. Instagram is a digital
social media platform for sharing photos and videos that can be edited using filters, hashtags, etc. Instagram has a hashtag feature that makes it easier to access by using the same hashtag. This has the
potential to make it easier to expand the reach of health content, especially quarter life crisis content. Instagram Reels can be an effective health education medium for providing information about the
quarter life crisis health. Reels is a feature on Instagram to allow users to create and display short to
long videos (Nur Mistari, 2023). Instagram Reels in this research was used to explain the quarter life
crisis that occurs in students and efforts to overcome the problem. p
The author's content is video reels with a duration of 6 minutes which begins with a scene of 2
female students who are experiencing a quarter life crisis in their identity and life goals. Followed by
interviews with several students and there were efforts to overcome the quarter life crisis. The
author's video has its own interest among Instagram users, as evidenced by the author's video views
reaching 3,786 and video likes reaching 93 likes. This shows that Instagram users gain knowledge
about the quarter life crisis. Not only from the number of views, Instagram users gave positive
feedback to the @phoffeedu Instagram account in the form of 31 comments. The average number of
comments in the video shows that listeners really accept the message conveyed in the video. Instagram users who view video content that has been presented not only do not benefit themselves,
but also provide it to other public audiences. This is proven by the number of shares in this post as
many as 9. This shows that the use of Instagram as social media for quarter life crisis education is
effective and has an impact. Inovasi Lokal Chris Heuer states that there are 4 indicators of the 4C theory regarding how to use social media
to have an effective impact, namely by using context, communication, collaboration and connection
(Luthfi et al., 2023). The communication strategy carried out by the Instagram account @phoffeedu
is to increase knowledge about health problems, especially the quarter life crisis topic with educative
and informative content with educative and informative content that utilizes the features provided by
Instagram (Tulandi, 2021). The first element, namely context, Instagram social media @phoffeedu can be called effective
because it has compiled, packaged and used language that is polite and easy to understand by
listeners, and is able to meet the needs of followers regarding the quarter life crisis. The Instagram
account @phoffeedu aims to increase knowledge of quarter life crises, as well as prevention efforts
in quarter life crises. To achieve this goal, the @phoffeedu Instagram account targets an audience
aged 18 to 30 years, at that age people often experience a quarter life crisis. The second element is communication, communication carried out by the Instagram account
@phoffeedu. The communication used is formal language so that readers can easily understand. The
message contained in the content is easily conveyed concisely. Content uploaded on Instagram shows
credibility because it includes existing sources. This is an open access article under Creative Commons
Attribution-ShareAlike 4.0 International License MATERIALS AND METHODS The implementation method for using social media uses qualitative methods, which include collecting
data by interviews and delivering material. During data collection, the author interviewed FIK UM
students about the Quarter Life Crisis. Then, delivery of material regarding efforts to overcome the
Quarter Life Crisis. The social media that we use to utilize social media is Instagram on the
@phoffeedu account. After uploading the video, the author examines the comments on the upload
and finds out whether the video can provide learning to students. Results and Discussion
Insight
Account Reach
690 Account Reached
Video Played
3.786 Plays
Likes
93 Likes
Comments
31 comments
Shares
9 shares
Saves
5 Saves Results and Discussion The existence of technology has an impact on all users, including teenagers. The increasing
number of Generation Z (Gen Z) children being born means that many people are using social media
for all their needs. In the future, the use of social media will become increasingly popular and all
people in the world will definitely not be separated from their daily activities. The percentage of the The existence of technology has an impact on all users, including teenagers. The increasing
number of Generation Z (Gen Z) children being born means that many people are using social media
for all their needs. In the future, the use of social media will become increasingly popular and all
people in the world will definitely not be separated from their daily activities. The percentage of the 137 Acknowledgement The author would like to express his thanks to all the help from the author who has designed a
video campaign on Instagram social media and created it in an article entitled "Using Social Media
as a Quarter Life Crisis Educational Media". Don't forget to say thank you to Instagram users who
have provided positive feedback to the @phoffeedu Instagram account in the form of likes and
comments. The comments seen in the video show that listeners really accept the message conveyed
in the video. Instagram users who see the video content that has been presented are not only useful
for themselves, but also for other public audiences. No Conflict of Interest Inovasi Lokal Pemberdayaan Masyarakat dalam Pembangunan Berkelanjutan Pemberdayaan Masyarakat dalam Pembangunan Berkelanjutan
Nusa Inovasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023 one of the biggest impacts that can make students or Gen Z feel a quarter life crisis. From the results
of making a video about the quarter life crisis by conducting interviews with students to get various
perspectives on the quarter life crisis and strategies for dealing with it, the video content will be
disseminated via social media such as Instagram. With the aim of taking an approach involving visual
narratives, interviews and practical solutions in providing a comprehensive view of the quarter life
crisis and providing positive value in using social media and having a good impact on yourself. CONCLUSIONS Quarter Life Crisis is an individual's response to moving towards the reality of life in which there is
instability, continuous change and the emergence of panic because they feel helpless. Currently, there
are still students who experience changes in emotional reactions such as anxiety, panic, stress,
confusion and doubt about their own abilities. Bad use of social media by viewing content that can
create a crisis of self-confidence, competition for a luxurious life and not wanting to accept reality is 138 Inovasi Lokal This is an open access article under Creative Commons
Attribution-ShareAlike 4.0 International License REFERENCES Ayu Khoirotul Umaroh, Fajrin, R., Kusumawati, M. A., Muhadzib, M. A., Haryudha, & Elisabet, B. M. (2023). Pemanfaatan Instagram sebagai Sumber Informasi Kesehatan Reproduksi Remaja (Studi
Kasus Akun @Tabu.id dengan Use and Gratification Theory). Media Publikasi Promosi
Kesehatan Indonesia (MPPKI), 6(1), 122–129. https://doi.org/10.56338/mppki.v6i1.2944 Fitriani, Y. (2021). Pemanfaatan Media Sosial Sebagai Media Penyajian Konten Edukasi atau
Pembelajaran Digital. Journal of Information System, Applied, Management, Accounting and
Research, 5(4), 1006–1013. https://doi.org/10.52362/jisamar.v5i4.609 Luthfi, M., Mubarak, M. T., Studi, P., Komunikasi, I., Gontor, U. D., & Hijrah, D. (2023). Efektivitas
Instagram Sebagai Media Informasi Pondok Modern Darul Hijrah Putra Martapura. 5, 161–179. Muttaqien, F., & Hidayati, F. (2020). Hubungan self efficacy dengan quarter life crisis pada mahasiswa
Fakultas Psikologi UIN Maulana Malik Ibrahim Malang angkatan 2015. … Jurnal Psikologi, 05, 75– Luthfi, M., Mubarak, M. T., Studi, P., Komunikasi, I., Gontor, U. D., & Hijrah, D. (2023). Efektivitas
Instagram Sebagai Media Informasi Pondok Modern Darul Hijrah Putra Martapura. 5, 161–179. Muttaqien, F., & Hidayati, F. (2020). Hubungan self efficacy dengan quarter life crisis pada mahasiswa
Fakultas Psikologi UIN Maulana Malik Ibrahim Malang angkatan 2015. … Jurnal Psikologi, 05, 75–
84. http://repository.uin-malang.ac.id/8383/%0Ahttp://repository.uin-
malang.ac.id/8383/1/8383.pdf Luthfi, M., Mubarak, M. T., Studi, P., Komunikasi, I., Gontor, U. D., & Hijrah, D. (2023). Efektivitas
Instagram Sebagai Media Informasi Pondok Modern Darul Hijrah Putra Martapura. 5, 161–179. Luthfi, M., Mubarak, M. T., Studi, P., Komunikasi, I., Gontor, U. D., & Hijrah, D. (2023). Efektivitas
Instagram Sebagai Media Informasi Pondok Modern Darul Hijrah Putra Martapura. 5, 161–179. Muttaqien, F., & Hidayati, F. (2020). Hubungan self efficacy dengan quarter life crisis pada mahasiswa Muttaqien, F., & Hidayati, F. (2020). Hubungan self efficacy dengan quarter life crisis pada mahasiswa Fakultas Psikologi UIN Maulana Malik Ibrahim Malang angkatan 2015. … Jurnal Psikologi, 05, 75–
84. http://repository.uin-malang.ac.id/8383/%0Ahttp://repository.uin-
malang.ac.id/8383/1/8383.pdf Nur Mistari, R. R. (2023). Pemanfaatan Media Sosial sebagai Media Penyajian Konten Edukasi
Stunting untuk Ibu Hamil. 7, 1276–1290. 139 This is an open access article under Creative Commons
Attribution-ShareAlike 4.0 International License Inovasi Lokal novasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023 Inovasi Lokal: Keberdayaan Masyarakat dalam Pembangunan Berkelanjutan, Vol 1 Issue 2 2023 Permatasari. A., Marsa. Ammar. Mohammad., S. (2022). Dampak Media Sosial Dalam Quarter Life
Crisis Gen Z di Indonesia. Jurnal Ilmiah Indonesia p–ISSN: 2541-0849 e-ISSN: 2548-1398,
7(8.5.2017), 2003–2005. Salsabila, T. (2021). Pengaruh quarter life crisis terhadap kepercayaan diri mahasiswa psikologi UIN
Malang. skripsi Program Studi Psikologi S1 Fakultas Psikologi Universitas Islam Negeri Maulana
Malik
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Malang,
15. http://etheses.uin-malang.ac.id/28132/%0Ahttp://etheses.uin-
malang.ac.id/28132/9/16410137.pdf Tulandi, E. V. (2021). Strategi Komunikasi Akun Instagram UbahStigma Dalam Meningkatkan
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Attribution-ShareAlike 4.0 International License
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EXPERIMENTAL ECOSYSTEM NATURAL CAPITAL ACCOUNTS IN THE REPUBLIC OF GUINEA
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The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye 1.1 From national land cover to ecosystem accounting Figure 1. The natural regions of the Republic of Guinea IGN FI is participating in the implementation of an ecosystem-
based accounting of natural capital (ENCA) based on its land-use
maps produced as part of the Guinea Agroecological Zoning
Project (ZAEG) (Jaffrain et al., 2021). The project, carried out in
collaboration with the Guinean Ministry of Agriculture, aims to
provide a decision-making tool for the government by mapping
land use and land use dynamics over a 10-year period and
identifying and assessing agricultural potential. Within the
framework of this project, two land use maps were produced
(2005 and 2015) thanks to photointerpretation and remote
sensing work carried out on site by a national team of Guinean
technicians trained and supervised by IGN FI experts. The land
use data produced from recent and old satellite images are the
basis of the ecosystem accounting of natural capital developed by
Jean-Louis Weber (Babin & Weber, 2019). It is worth recalling
here that the Convention on Biological Diversity (CBD)
published a manual in 2014 to support countries in the
implementation of ENCA . Figure 1. The natural regions of the Republic of Guinea The Republic of Guinea (Fig.1), a West African country whose
capital Conakry is located on the Atlantic coast, is therefore the
territory chosen for our experimentation with Ecosystemic
Accounting of Natural Capital. ABSTRACT : Over the past thirty years (Rio Conference, 1992), the climate has become an important issue in world politics. With the Kyoto Protocol
(1997), atmospheric carbon accounting has gradually been introduced with the aim of raising awareness of national and international
decision-making systems for the implementation of an energy transition. However, this carbon approach does not take into account
ecosystems despite their fundamental role in climate regulation. A global assessment of all natural resources and ecosystem services,
known as natural capital, is necessary from a sustainable development perspective. This assessment must then be taken into account in
national accounting systems. The aim of this article is to test, on the national territory of the Republic of Guinea, the ecosystem-based
natural capital accounting method developed by Jean-Louis Weber (Weber, 2014). Based on three accounts (ecosystem infrastructure,
ecosystem carbon and water resources), this method aims to measure the sustainable capacity or 'sustainability' of ecosystems to
provide services. Based on land use & land cover layers produced in the framework of the agroecological zoning project (Jaffrain et
al., 2021), we have operationalised this ecosystem accounting methodology in the Republic of Guinea to calculate the total
sustainability of the ecosystem. The land cover layers are the basic structural data for monitoring and describing the evolution of the
territory at different temporal intervals. Thus, several environmental indicators were defined from these combined geospatial data and
eventually allowed to define the evolution of the total sustainability of the territory's ecosystem between 2005 and 2015. A clear
degradation of this sustainability value was identified, which reflects the numerous land use changes affecting the country in the recent
period (2005-2015). EXPERIMENTAL ECOSYSTEM NATURAL CAPITAL ACCOUNTS
IN THE REPUBLIC OF GUINEA J.-A. Morand 1, G. Jaffrain 1, C. Sannier 1, J.-L. Weber 2
1 IGN FI, Paris - Bastille, France - (jamorand, gjaffrain, csannier)@ignfi.fr
2 Independent consultant of ENCA - jlweber45@gmx.fr J.-A. Morand 1, G. Jaffrain 1, C. Sannier 1, J.-L. Weber 2 1 IGN FI, Paris - Bastille, France - (jamorand, gjaffrain, csannier)@ignfi.fr
2 Independent consultant of ENCA - jlweber45@gmx.fr KEY WORDS: remote sensing, ecosystem accounting, environmental indicator, landcover, landcover change, carbon, water,
watershed, Guinea 1.
INTRODUCTION years by numerous changes in land use (artificialization,
agricultural expansion, major development projects, etc.). This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. This contribution has been peer-reviewed. 2.1 Accounting tool The economic performance of societies is one of the main
priorities of governments worldwide. The economic activities of
human societies are highly dependent on the services provided
by ecosystems. Yet the "natural capital" that refers to ecosystems
is not taken into account in national accounting. This "oversight"
on the part of national decision-makers is one of the main factors
behind the decline in biodiversity. To counteract this trend,
initiatives have been launched since the end of the 20th century
to develop accounting systems that combine environmental and
economic dimensions. The term natural capital, at the crossroads
of ecology and economics, is defined as "the natural wealth that
provides society with renewable and non-renewable resources
and ecosystem services" (Ten Brink, 2016). The need to integrate natural ecosystems into international
economic exchanges and policies has been apparent for several
years. As early as 1992, the Convention on Biological Diversity
(CBD), signed in Rio de Janeiro, expressed concern about the
rapid degradation of biodiversity and set a number of main
objectives: "the conservation of the various forms of life, the
sustainable use of its components so as not to jeopardise the
renewal capacity of natural environments and access to genetic
resources, as well as the fair sharing of the benefits arising from
their use" (Lévêque & Duhautois, 2006). In 2010, within the
framework of the United Nations Convention on Biological
Diversity in Nagoya, Japan, a Strategic Plan for Biological
Diversity 2011-2020 was adopted. In it, 20 biodiversity targets
called Aichi targets are established and organised into 5 strategic
goals. In order to take ecosystems into account within natural capital,
multiple measurement and quantification approaches are being
developed. Among these tools, the Artificial Intelligence For
Ecosystem Services (ARIES) developed by the University of
Vermont makes it possible to map and quantify ecosystem
services and their beneficiaries at the scale of a territory
(IONESCU et al., 2019). Another example is the Integrated
Valuation of Ecosystem Services and Tradeoffs (InVEST), a
suite of software models based on ecosystem services. These
models are designed for different types of ecosystems (terrestrial,
aquatic, coastal ...). Governmental" natural capital accounting tools account for the
variation and evolution of ecosystems at the scale of an entire
territory. 2.1 Accounting tool These integrated accounts seek to aggregate biophysical
and socio-economic information in order to guide and provide a
decision-making tool for public policies on ecosystems and also
to inform the population on the ecosystem services provided by
a territory (Feger & Mermet, 2021). The integration of "biological diversity throughout government
and society" (Convention on Biological Diversity & UNEP,
2010) is at the heart of the first strategic goal. The second Aichi
Goal proposes to integrate biodiversity "into national and local
development strategies and planning processes" and to
incorporate it "into national accounts" (Convention on Biological
Diversity & UNEP, 2010). In order to keep up with the evolution
of society and respond to the challenges posed by climate change,
the Sustainable Development Goals (SDGs) were adopted by the
United Nations General Assembly in September 2015. 17 SDGs
are set out in the 2030 Agenda for Sustainable Development,
covering all development issues (climate, biodiversity, energy,
agriculture, etc.). Initiated in 2003 by the feasibility study on land and ecosystem
accounts conducted by the European Environment Agency (J.-L. Weber & European Environment Agency, 2006), the ENCA
approach developed by Jean-Louis Weber is in line with a
perspective that favours the safeguarding of natural capital. The
ENCA quick Start Package published by the Convention on
Biological Diversity in 2014 (Weber, 2014) to assist countries
wishing to carry out ecosystem accounting. Thus, SDG 15.9 (target 9), inspired by the second Aïchi Goal,
deals with the integration of "the protection of ecosystems and
biodiversity into national planning, development mechanisms
and accounting"(United Nations, 2015). SDG 17.19 (target 19)
leads to a reflection on the construction of indicators of progress
in sustainable development, enriching the Gross Domestic
Product (GDP) and strengthening the statistical capacities of
developing countries (Babin & Weber, 2019). In this context,
ENCA, developed by Jean-Louis Weber, is an approach that fits
perfectly into the global political framework by responding to
several Sustainable Development Goals such as the Convention
on Biodiversity (SDG 15.9), the Convention on Combating
Desertification and Land Degradation Neutrality (SDG 15.3) and
the 2015 Paris Agreement on Climate Change... Indeed, ENCA
appears to be a decision-making tool for countries in terms of
environmental protection and management. In addition, the approach is recognised by the United Nations
Statistical Commission as fitting within the broad ecosystem
accounting framework of the System of Environmental and
Economic Accounts (SEEA). 2. DATA AND METHODS change. Since then, a climate accounting system based on the
guidelines of the Intergovernmental Panel on Climate Change
(IPCC) has been set up around a unit of measurement: the CO2
equivalent. However, this accounting does not take into account
the functions of ecosystems or their role in regulating the climate. Ecosystems are therefore only considered as carbon stocks, even
though they are important assets in adapting to climate change,
since they are at the origin of biological, biophysical and
biogeochemical processes. Their state of health is not taken into
account in the wealth of States (J.-L. Weber, 2022). However, the
degradation of ecosystems (reduction in biodiversity, reduction
in sequestration functions, etc.), as well as the emission of
greenhouse gases, is also one of the causes of global warming
(Delangue & Teillac-Deschamp, 2019). 1.2 Accounting in line with the Sustainable Development
Goals (SDGs) Since the end of the 20th century, climate has become an
important policy issue worldwide. Numerous scientific studies
point to climate change and a rapid decline in biodiversity. In
1997, at the third Conference of the Parties (COP 3) of the United
Nations Framework Convention on Climate Change (UNFCCC),
the Kyoto Protocol committed to a global policy to reduce
greenhouse gas emissions, as the increase of carbon dioxide
(CO2) in the atmosphere is one of the main causes of climate With a surface area of 245,857 km², the country is subdivided
into four large geographical zones, otherwise known as natural
regions: Lower Guinea (coastal zone), Middle Guinea
(mountainous zone), Upper Guinea (savannah zone located in the
north-east) and Forest Guinea (dense rainforest zone). With a rapidly growing population and the largest bauxite and
iron reserves in the world, its territory has been marked in recent 203 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye 2.1 Accounting tool ENCA is a comprehensive
approach to integrating and synthesising biophysical and socio-
economic data on sustainability and potential across all
ecosystems (continental, coastal, natural, man-made...) in a
territory or country. It aims to measure the capacity of
ecosystems to provide services (ecosystem potential) both in the
short term and in the future. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. This contribution has been peer-reviewed. 2.2 General methodology ENCA This ecosystem capacity of ecosystem capital is calculated, at a
given date for a defined area, on the basis of three accounts:
ecosystem carbon, water resources and ecosystem infrastructure
(biodiversity and river, land infrastructure, etc.). In the course of this work, it was therefore necessary to carry out
a first experimentation of ENCA on the territory of the Republic
of Guinea. The main accounting methods are reviewed, and the
methodology of ecosystem accounting is then detailed. Finally,
the first results of the application of this method in this West
African territory are presented, accompanied by maps. The stocks and biophysical flows of natural capital are compared
between two dates to better understand their evolution over time
'resulting from natural renewal and resource use flows' (Babin &
Weber, 2019) (see Fig. 2 below). 204 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye national mapping agencies or ministries, such as those in charge
of spatial planning (Weber, 2014). Figure 3. SELU cover the Républic of Guinea
(HydroBASINS, Lehner & Grill, 2013) national mapping agencies or ministries, such as those in charge
of spatial planning (Weber, 2014). ENCA therefore consists of an inventory and then a diagnosis of
the degradation or improvement of ecosystems by establishing
biophysical and ecological balances on geo-referenced land use
bases. national mapping agencies or ministries, such as those in charge
of spatial planning (Weber, 2014). ENCA therefore consists of an inventory and then a diagnosis of
the degradation or improvement of ecosystems by establishing
biophysical and ecological balances on geo-referenced land use
bases. Figure 2. Overview of the Ecosystem Natural Capital
Accounting (ENCA) Framework Figure 3. SELU cover the Républic of Guinea
(HydroBASINS, Lehner & Grill, 2013) Figure 2. Overview of the Ecosystem Natural Capital
Accounting (ENCA) Framework Examples include watershed data available in HydroBASINS or
river
maps
obtained
from
WWF-HydroSHEDS/FAO-
AQUASTAT sources. 2.3 A robust infrastructure The basic functional unit of ENCA is the Socio-Ecological
Landscape Unit (SELU). It is a spatialized unit (Fig. 3) built
around the combination of two dimensions: the dominant
landscape type and the belonging to a watershed. It thus
integrates an essential geographical element (the catchment area)
and makes it possible to describe various variables such as the
water resource and its accessibility on a territory. In order to
produce it, the start-up manual of the Convention on Biological
Diversity (Weber, 2014) proposes several methods. The one
chosen for the Republic of Guinea consists in using the small
river basins as the basic boundary of the units and assigning to
them the dominant landscape type of land cover. 2.2 General methodology ENCA Socio-economic statistical data are used in
the calculation of ENCA, notably national agricultural statistics
(FAOSTAT). Thus, apart from the land use maps produced by
IGN FI, all the data used come from global and national open
access databases. As with carbon accounting and the CO2 equivalent unit, the aim
is to establish an Ecosystem Capability Unit (ECU) to assess the
sustainability of ecosystems. This ECU measure should, among
other things, make it possible to measure the resource that is
accessible, i.e. that can be used without causing ecosystem
degradation. Subsequently, ENCA plans to transform this ECU
into a monetary value based on the costs of protection and
restoration, among other things (Weber, 2022). The resilience of
ecosystems is therefore at the heart of this approach, which is in
line with a strong sustainability trend. Depending on the availability of the data, the scale of the
information is adapted. Thus, for local applications, more precise
monitoring data or even field observation data can be integrated
to enrich the calculation of the accounts. If the data is not
available on a local/regional scale, the use of national and
international databases is preferred. The latter are constantly
being improved and updates are to be expected over time,
particularly with the availability of regularly updated high-
resolution satellite images (Sentinel, Landsat 8, SPOT, etc.) and
the development of more efficient monitoring systems. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. 2.4 Land accounts : the foundation for the account Geographic reference layers (administrative
boundaries, road networks, etc.) are easily accessible online via 205 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye In order to complete this account of the biophysical integrity of
ecosystems, a table dealing with their state of health is included. This includes, among others, an index of local biodiversity
integrity, the Local Biodiversity Intactness Index (Local
Biodiversity Intactness Index – GEO BON, s. d.), which
estimates the proportion of biodiversity of a terrestrial
environment in relation to human use. The interpretation of land cover classification changes allows the
identification of the processes at their origin and thus to group
them into fluxes. A land cover flux is thus 'a grouping of changes
of the same nature' (Jaffrain et al., 2021) which allows a better
understanding of the changes that have occurred by integrating
information on the land cover prior to the change. These main
flows can then be detailed at finer sub-levels according to the
specificities of the regional context. This requires a land cover
classification also detailed at different levels. The realization of
the land account already allows the characterization of some
indicators such as deforestation, artificialisation, etc. The overall access to these functional ecosystem services is based
on the proximity between the ecosystem infrastructure and
people. A series of indicators (local access of the population to
the TEIP, access to water regulation services, etc.) is grouped
together in another accounting table. These indicators combine
the different potentials of the ecosystem infrastructure with
demographic data (Babin & Weber, 2019). The ENCA method combines qualitative (e.g. ecosystem health
indicators) and quantitative (e.g. soil carbon content in tonnes)
measures on three basic accounting balances: biocarbon, water
and ecosystem infrastructure. These accounts are built around
land cover as a foundation. The combination of these accounts
makes it possible to measure the total ecosystem capacity of
natural capital (unit ecological value) in order to gauge its
evolution (improvement or degradation) over a given territory
and time interval. 2.6 Ecosystem carbon account The ecosystem carbon account records carbon stocks and flows. It aims to assess the sustainable capacity of ecosystems to
produce biomass (measured in biocarbon). The evolution of this
biomass value is also taken into account through various
biophysical processes: agriculture and crops, soil erosion, forest
fires, etc. This account therefore deals with the entire carbon
cycle, with the exception of fossil carbon resources. As with the
ecosystem infrastructure account, each element of the carbon
cycle is considered in structured tables. For the time being, only
carbon stored in the biosphere has been considered. Atmospheric
carbon will be considered in an additional table at a later stage. 2.5 Ecosystem infrastructure account According
to
the
Millennium
Ecosystem
Assessment
(Millennium
Ecosystem
Assessment
(Program),
2005),
ecosystems are multifunctional, providing a range of both
tangible and directly measurable services (e.g. provisioning
services) and intangible services (e.g. cultural and regulatory
services). In ENCA, the latter are included in the ecosystem
account of functional landscapes (also called "ecosystem
infrastructure") which measures the capacity of ecosystems to
provide these intangible services. It includes the services of
landscapes, rivers and coastal marine areas to provide services
that cannot be directly measured as physical quantities. The
ecosystem infrastructure account describes changes in stocks
between two dates. Its balance sheet is structured in four tables. It focuses first on a quantitative description of the extent of
ecosystems with the calculation of stock, land cover in hectares,
and a balance of rivers in terms of linear lengths. First, carbon stocks are recorded for vegetation (living above-
ground biomass), litter and roots and soil organic carbon. Forestry data from ESACCI (ESA Climate Change Initiative)
and Hansen (University of Maryland) are used to estimate forest
biomass at both dates. The International Soil Reference and Information Centre (ISRIC)
provides soil biocarbon data per hectare at different depths. Terrestrial animal biocarbon is also taken into account, starting
with livestock (FAO data). All data produced is converted to
tonnes of carbon using the equivalence of 50% biocarbon in
biomass. The account then describes biocarbon flows within ecosystems. Inputs are mainly based on net primary production (NPP) data. Biocarbon outputs include agricultural and forestry harvests (tree
felling), soil erosion, losses due to land use changes (such as
artificialisation), wood burning, forest fires. FAO data and
national statistics are used to determine agricultural crops. The account then describes biocarbon flows within ecosystems. Inputs are mainly based on net primary production (NPP) data. Biocarbon outputs include agricultural and forestry harvests (tree
felling), soil erosion, losses due to land use changes (such as
artificialisation), wood burning, forest fires. FAO data and
national statistics are used to determine agricultural crops. Forest biomass stocks, previously calculated for carbon stocks,
are used to estimate the extraction of roundwood from the forest
and the residues of forestry (dead leaves, branches, bark...). In ENCA, carbon inputs are also taken into account in the "input"
flows. Indeed, part of the biocarbon used by human activities
returns to the ecosystem (production returns): crop residues,
forestry residues, livestock manure. 2.4 Land accounts : the foundation for the account The integrated nature of the accounting
framework makes it possible to calculate, for each of the thematic
accounts, an index of sustainable use and an index of resilience
or health. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. 2.4 Land accounts : the foundation for the account In the ENCA system, land cover data are central, as they reflect
biophysical characteristics (vegetation cover, density, height...)
and land use. According to the FAO, land cover indicates the
physical cover of the land such as forests and wetlands. Land use
indicates the use of the land cover by humans activities (such as
crops or fallow land). Within the framework of the Guinea agro-ecological zoning
project, land use maps are produced based on the CORINE Land
Cover nomenclature (Jaffrain in Feranec, European landscapes
dynamics, 2016) and integrating modifications linked to regional
specificities, notably by relying on the Yangambi classification
(Aubréville, 1957). Two land coverages were carried out by
remote sensing and photointerpretation for the years 2005 and
2015. SPOT 4/5 and SPOT 6/7 satellite data were used for 2005
and 2015, respectively, over the entire territory of Guinea
(Jaffrain et al., 2021). Figure 3 shows the socio-ecological landscape units selected for
Guinea. These are based on the Hydroshed 10 (HYBAS 10) level
retrieved from the HydroBASINS database which is composed
of a series of vectorised polygon layers that follow the boundaries
of sub-catchments of different sizes (Lehner & Grill, 2013). ENCA requires a large amount of collected data (freely available
online) with different resolutions (from 10-30 m to 250 m). A
spatial resolution of 100 m by 100 m was chosen over the
territory of the Republic of Guinea. As a result, all geographic data are resampled to a 1 ha grid
corresponding to the project grid. A common projection system
is also chosen for the project, which may lead to a reprojection of
some geographical data. SAGA software (Conrad et al., SAGA,
2015) is used to carry out most of the ENCA process. The land account based on information from the land cover
databases provides a first assessment of land cover changes over
a period of time. It is the basic account required for the
construction of the ENCA. Based on the gains and losses of areas
(in ha) between two dates, it first allows the establishment of a
matrix of changes in terms of land cover classes. This matrix is
produced from the SAGA GIS software. ENCA is produced from various types of national and
international data. 3. RESULTATS As explained above, the years 2005 and 2015, for which land
cover data are available, were selected for the implementation of
the ENCA for the Republic of Guinea. All indicators were
therefore calculated for both dates. For the sake of simplicity,
only a few relevant and representative indicators for the territory
dealing with 2015 data appear in this section. 2.7 Water accounts Water accounts (especially in the form of water balance) are
commonly used in hydrology and agronomy around the world. As early as the 1980s, accounts of water quantities per catchment
area were produced in France and Spain, taking into account both
the volume and the quality of the water. These experiences allow
the SEEA to integrate, in 2007, a sub-system of economic and
environmental water accounting focusing on the use of water by
the economy. In ENCA, the objective is to diagnose the
components of the ecosystem and thus to assess the impact of a
degradation of the water resource on the whole ecosystem. The
ecosystem water account is therefore an extension to the SEEA
water accounts (Weber, 2014). 3.1 Some indicators for the infrastructure account The Net Landscapes Ecosystem Potential (NLEP) is composed
of the Green Background Landscape Index (GBLI). This
estimates the naturally sustainable biomass of various land cover
types. For example, natural forests with little anthropogenic
activity and wetlands have a high index, whereas a large-scale
monoculture, because of its dependence on human inputs (seeds,
fertilisers, etc.), has a low GBLI. In order to calculate this index,
a score from 0 to 100 is assigned to the different land cover
classes. To improve the GBLI, we combined it with a forest
density index produced by the University of Maryland (Hansen
data). By averaging these two indices, a synthetic index (see Fig. 4 below) is obtained that is more representative of changes in the
natural landscape and considers density variations within classes
(especially in mixed landscapes). The ENCA water account addresses all the interactions and
exchanges of the water cycle in a territory by recording the
different stocks and flows. The hydrological system is treated as
a whole as well as the flows of water use by human activities. To
begin with, the basic ecosystem water balance records the water
stocks of lakes, rivers, soil and vegetation. In the infrastructure
account, rivers are treated only in terms of their accessibility
through the River Accessibility Weighted Index (RAWI). However, in both the infrastructure and water accounts, rivers are
calculated in terms of their potential, not just their volume of
water. The Standard River Measurement Unit (SRMU) allows for
the length as well as the flow of rivers. A single unit of flow can
only provide information at a single point on the river, whereas a
river is a continuum of points. The concept of a "standardized
river-kilometer" was suggested (Heldal & Østdahl, 1984). Figure 4. Map of Green background landscape Indicator
(GBLI) per SELU in 2005 It is defined as a 1 km long stretch of watercourse (river, stream)
that has a flow of 1 m³/s at any point along its course. The basic
balance then describes the inflow and outflow of rivers and
groundwater
following
the
series
"precipitation,
evapotranspiration, infiltration and runoff" (Weber, 2021). Data are mainly taken from global meteorological databases
(such as WorldClim, n.d.) but local monitoring stations can also
be used. 2.5 Ecosystem infrastructure account In particular, an
index of the stability of carbon pools in forest ecosystems and an 206 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye index of soil resistance to erosion are calculated in the
implementation done on the Republic of Guinea. index of soil resistance to erosion are calculated in the
implementation done on the Republic of Guinea. index of soil resistance to erosion are calculated in the
implementation done on the Republic of Guinea. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. 2.5 Ecosystem infrastructure account The carbon content of
manure is estimated on the basis of livestock units (LU). The potential of the ecosystem infrastructure to provide services
is measured by a combination of several types of environmental
indicators. On the one hand, indicators on the biophysical
characteristics of the ecosystem infrastructure and on the other
hand indicators on the health of the ecosystem. The first type of indicator consists of the Net Landscapes
Ecosystem Potential (NLEP) and Rivers (NREP). These two
composite indicators are classified and take into account an index
of sustainability of terrestrial and river environments, based on
land use and forest density in the case of the PENP. Combined
with this, an index specifying the natural value of the territory
based on the status of natural protection (wildlife reserves,
national parks, etc.) extracted from national and international
maps. Also taken into consideration is the degree of artificial
fragmentation of the territory linked to road and rail
infrastructures and dams (on rivers), which at a certain level
limits exchanges between ecosystems (reduces the movement of
species and the connectivity of habitats). The NLEP and NREP
are finally aggregated into the Total Ecosystem Infrastructure
Potential (TEIP). Thus, the ENCA ecosystem carbon account starts with a baseline
ecosystem carbon balance (total inputs) and then records the
accessible resource, i.e. the measure of ecosystem carbon that can
be used in a sustainable manner. Thereafter, the ENCA method deals with total ecosystem
biocarbon use presented as the sum of total removals and indirect
anthropogenic net losses of biocarbon due to land use (such as
artificialisation). The accounting concludes with a table of
indices of use intensities and ecosystem health. 3.2 Some indicators of carbon accounts 3.2 Some indicators of carbon accounts Figure 5. Map of the Landscape High Nature Value Index
(LHNVI) by UPSE Figure 5. Map of the Landscape High Nature Value Index
(LHNVI) by UPSE Figure 7. Living above-ground biomass in 2015 (t/ha) According to the maps produced (Fig. 5), Forest Guinea, a natural
region in the south-east of the country, has a high green landscape
background index. This could be correlated with the existence of
several protection zones. However, this correlation between the
GBLI and LHNVI indices does not necessarily hold true for the
whole country. The main causes of variation in ecosystem carbon stocks are
variations in above-ground biomass stocks (living biomass and
litter, dead wood) and soil organic carbon (also includes living
roots, litter and dead wood). In the Republic of Guinea, the levels of tree biomass stocks are
particularly high in the natural region of Forest Guinea. Up to 200 t/ha of tree biomass (Fig. 7) are observed in the
classified forest of the Ziama massif, designated as a biosphere
reserve in 1980 by UNESCO (The Ziama Massif: a vestige of the
decline of forest ecosystems in High Guinea, West Africa, s. d.) In ENCA, the landscape fragmentation index is used to adjust the
green and high nature value landscape fund indices by taking into
account the barrier effects that can limit interactions between
ecosystems. Produced from maps of the main road and rail
networks, it represents the "strong" fragmentation of the
landscape, i.e. that which induces significant negative effects
(isolation of populations, disruption of trophic chains, etc.) on the
ecosystems. Thus, the landscape fragmentation index reflects the
degree of fragmentation of natural (non-artificial) land cover
areas. An unfragmented landscape unit (SELU) has a value of 1. The lower the mesh value, the more fragmented the natural areas
within the unit are. On the map below (Fig. 6), a low mesh size
value and therefore a high fragmentation index (red on the map)
can be observed in SELUs with dense transport infrastructures,
such as the capital, Conakry. From this biomass value in tons is estimated the biocarbon stock
(Figure 8) using the ratio of ½ of biocarbon in the biomass. Figure 8. Carbon in living above-ground biomass in 2015 (t/ha) Figure 6. Map of the Landscape Fragmentation Indicator, by
SELU –data in 2005 Figure 8. 3.1 Some indicators for the infrastructure account Natural inflows and outflows from HYBAS 10
catchments (on which the SELU is based) are measured, as well
as water flows related to irrigation and other uses (hydropower
dams, cooling water, etc.), based on national statistical data
where available. The water use account summarises all recorded
water uses. In order to assess the sustainability of the water
resource, it is important to know the accessible water resource,
i.e. the water whose use by humans does not endanger the
provision of the service by the ecosystem. Figure 4. Map of Green background landscape Indicator
(GBLI) per SELU in 2005 The Landscape High Nature Value Index (LHNVI) is based on
maps of protected areas. The aim is to get an indication of the
natural conservation value of the area concerned. Indeed, the
index should reflect the level of protection of the area. p
y
y
One of the final calculations in the water accounts is the net
accessible water surplus balance, which describes the exploitable
water resource, taking into account potential limitations on use
(Weber, 2014). Finally, as with the other accounts, the water
accounts end with a table on the intensity of use index, which
specifies the degradation (below 1) or not of ecosystems. It is
defined by the ratio of the exploitable water resource of the
ecosystem to the total water use. In addition to this, a composite
indicator on the change in ecosystem water health (including
biochemical water quality) is calculated (Babin & Weber, 2019). At the end of each account (water, carbon, infrastructure) the
internal ecological unit value per SELU is calculated by
combining these two indicators, a health status indicator, and a
change indicator. This value calculated for each ecosystem
component reflects its integrity, health and resilience. Similarly, where an area has more than one type of protection, it
is assumed that it has a higher value and therefore the protections
add up. An area that is not protected or not designated for its high
nature value has a value of 1. In this way, one level of protection provides a score of 2, and so
on. In the case of Guinea, there are 3 levels of protection as set
out below: - Level 1: Unprotected areas - Level 2: Sacred forests; Wildlife reserves - Level 3: Classified forests; Strict reserves, National parks This contribution has been peer-reviewed. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. This contribution has been peer-reviewed. 3.1 Some indicators for the infrastructure account 207 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye 3.2 Some indicators of carbon accounts
Figure 7. Living above-ground biomass in 2015 (t/ha) 3.2 Some indicators of carbon accounts Carbon in living above-ground biomass in 2015 (t/ha) Based on data from the International Soil Reference and
Information Centre (ISRIC), the soil organic carbon stock is
essentially the result of residues of plant and animal matter,
decomposed under the influence of temperature, humidity,
environmental conditions, and soil micro-organisms. Soil organic carbon is heterogeneously distributed over the
Guinean territory. Figure 6. Map of the Landscape Fragmentation Indicator, by
SELU –data in 2005 By cross-referencing this map with the land use layer, it can be
seen that the coastal mangrove areas have the highest carbon rates
per hectare (300 to 400t/ha). Land use changes, particularly agricultural expansion dynamics
in these areas, should therefore have an impact on soil organic
carbon stocks. 208 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
39th International Symposium on Remote Sensing of Environment (ISRSE-39) “From Human Needs to SDGs”, 24–28 April 2023, Antalya, Türkiye Figure 9. Soil Carbon at 1m depth (T/ha) 3.4 Final summary of ecosystem potentials From the table below (Fig.12), we see a decrease in the total
ecosystem potential at the scale of Guinea. The value went from
1.03.109 to 1.01.109. This variation can be explained by the
numerous land use and land cover changes that took place in the
territory between 2005 and 2015. Figure 9. Soil Carbon at 1m depth (T/ha) One of the main biocarbon input streams into the system is the
net primary production of ecosystems, which is based on the
overall dry matter productivity directly related to the growth rates
of vegetation biomass. Net primary productivity is higher in 2015
compared to 2005. Figure 12 : Total ecosystem capability (TEC) of Guinea for the
years 2005 and 2015
2005
2015
Carbon
Ecosystem
Capability
C_EC_capab
1,56E+08
2,03E+08
Water Ecosystem
Capability
W_EC_capa
b
1,23E+08
1,39E+08
Ecosystem
Infrastructure
Capability
EI_EC_capa
b
7,49E+08
6,67E+08
Total Ecosystem
Capability
TEC_capabi
lity
1,03E+09
1,01E+09
Years
Potentials Figure 12 : Total ecosystem capability (TEC) of Guinea for the
years 2005 and 2015
2005
2015
Carbon
Ecosystem
Capability
C_EC_capab
1,56E+08
2,03E+08
Water Ecosystem
Capability
W_EC_capa
b
1,23E+08
1,39E+08
Ecosystem
Infrastructure
Capability
EI_EC_capa
b
7,49E+08
6,67E+08
Total Ecosystem
Capability
TEC_capabi
lity
1,03E+09
1,01E+09
Years
Potentials Figure 10. Net Primary Productivity (NPP), in tonnes of carbon
per ha, by UPSE – data in 2015 Figure 12 : Total ecosystem capability (TEC) of Guinea for the
years 2005 and 2015 Figure 12 : Total ecosystem capability (TEC) of Guinea for the
years 2005 and 2015 Figure 10. Net Primary Productivity (NPP), in tonnes of carbon
per ha, by UPSE – data in 2015 4. DISCUSSION Ecosystem-based natural capital accounting allows the evolution
of the capacity of ecosystems to provide sustainable services to
be assessed. This inherent capacity is an integral part of
measuring the ecological value of each of the ecosystem
components. The land cover produced by the IGN FI teams
provided a perfect testing ground for the CECN at the scale of a
national territory. Figure 11. Average rainfall (mm) for 2005 (top) and 2015
(bottom) The main input to the water account is rainfall. An increase in
average annual rainfall in Guinea between 2005 and 2015 was
observed, which partly explains the increase in net primary
productivity (see Fig. 10) observed in the area. The distribution
of rainfall is heterogeneous and varies across the country (Fig. 11). The coast is wetter, while the north-east of the country is
drier. In order to smooth out interannual variations, 3-year
averages were used. p
https://doi.org/10.5194/isprs-archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. This contribution has been peer-reviewed. p
archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVIII-M-1-2023-203-2023 | © Author(s) 2023. CC BY 4.0 License. the problems of implementing such a method in the Republic of
Guinea. the problems of implementing such a method in the Republic of
Guinea. Photogrammétrie
et
de
Télédétection,
223,
59‑80. https://doi.org/10.52638/rfpt.2021.563 the problems of implementing such a method in the Republic of
Guinea. Le massif de Ziama : Vestige de la diminution de l’écosystème
forestier de Haute-Guinée | Afrique occidentale. (s. d.). Consulté
28 juillet 2022, à l’adresse https://eros.usgs.gov/westafrica/case-
study/ziama-massif-relic-diminishing-upper-guinean-forest-
ecosystem As the ENCA method depends on the data used, the latter play an
essential role in the smooth running of the method. The quality
and availability of the data for the periods studied (2005 and
2015) sometimes raise difficulties. Indeed, some data, such as the
OpenStreetMap road network map, are too heterogeneous and/or
undated, making the resulting indicator (of landscape
fragmentation) less representative of the reality on the ground
and more static (available only for one date). Lehner, B., & Grill, G., 2013. Global river hydrography and
network routing : Baseline data and new approaches to study the
world’s large river systems. Hydrological Processes, 27(15) :
2171–2186. www.hydrosheds.org The ENCA protocol is currently being improved, in particular to
better integrate the morphology of the territory and the relief in
the definition of socio-ecological landscape units (SELU), or to
better take into account marine coastal ecosystems in the
accounts. Lévêque, A., & Duhautois, L., 2006. L’environnement en France
(édition 2006). Chapitre « La biodiversité ». (p. 273‑296). Local Biodiversity Intactness Index – GEO BON. (s. d.). Accessed
on
July
19,
2022,
à
l’adresse
https://geobon.org/ebvs/indicators/local-biodiversity-intactness-
index/ 3.3 Some indicators of water accounts The ENCA water account is calculated from available input data
on stocks (lakes and reservoirs, rivers) and input and output flows
(rainfall, evapotranspiration, irrigation abstraction). The results of this work in Guinea already demonstrate the
importance of a policy to maintain the integrity of ecosystems
with regard to the degradation of the total ecosystem potential
linked to that of the internal unit value of the ecosystem
infrastructure. ENCA process thus appears to be an interesting decision-making
tool for countries on environmental issues. From an economic point of view, when this total capacity reflects
a degradation of the ecosystem, it leads to unpaid ecological costs
produced by the perpetrator(s) of the degradation. These
ecological debts could be measured to estimate the restoration
value of the degraded ecosystem (Babin & Weber, 2019). In
addition, this experimentation makes it possible to advance on This contribution has been peer-reviewed. 209 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVIII-M-1-2023
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Assignment of the somatic A/B compartments to chromatin domains in giant transcriptionally active lampbrush chromosomes
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Epigenetics & chromatin
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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Krasikova et al. Epigenetics & Chromatin (2023) 16:24
https://doi.org/10.1186/s13072-023-00499-2 Krasikova et al. Epigenetics & Chromatin (2023) 16:24
https://doi.org/10.1186/s13072-023-00499-2 Epigenetics & Chromatin Open Access Assignment of the somatic A/B
compartments to chromatin domains
in giant transcriptionally active lampbrush
chromosomes Alla Krasikova1*, Tatiana Kulikova1, Juan Sebastian Rodriguez Ramos1 and Antonina Maslova Abstract Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. *Correspondence:
Alla Krasikova
a.krasikova@spbu.ru
Full list of author information is available at the end of the article *Correspondence:
Alla Krasikova
a.krasikova@spbu.ru
Full list of author information is available at the end of the article *Correspondence:
Alla Krasikova
a.krasikova@spbu.ru
Full list of author information is available at the end of the article Abstract Background The three-dimensional configuration of the eukaryotic genome is an emerging area of research. Chro-
mosome conformation capture outlined genome segregation into large scale A and B compartments corresponding
mainly to transcriptionally active and repressive chromatin. It remains unknown how the compartmentalization of the
genome changes in growing oocytes of animals with hypertranscriptional type of oogenesis. Such oocytes are char-
acterized by highly elongated chromosomes, called lampbrush chromosomes, which acquire a typical chromomere-
loop appearance, representing one of the classical model systems for exploring the structural and functional organi-
zation of chromatin domains. Results Here, we compared the distribution of A/B compartments in chicken somatic cells with chromatin domains
in lampbrush chromosomes. We found that in lampbrush chromosomes, the extended chromatin domains, restricted
by compartment boundaries in somatic cells, disintegrate into individual chromomeres. Next, we performed FISH-
mapping of the genomic loci, which belong to A or B chromatin compartments as well as to A/B compartment transi-
tion regions in embryonic fibroblasts on isolated lampbrush chromosomes. We found, that in chicken lampbrush
chromosomes, clusters of dense compact chromomeres bearing short lateral loops and enriched with repressive epi-
genetic modifications generally correspond to constitutive B compartments in somatic cells. A compartments align
with lampbrush chromosome segments with smaller, less compact chromomeres, longer lateral loops, and a higher
transcriptional status. Clusters of small loose chromomeres with relatively long lateral loops show no obvious corre-
spondence with either A or B compartment identity. Some genes belonging to facultative B (sub-) compartments can
be tissue-specifically transcribed during oogenesis, forming distinct lateral loops. Conclusions Here, we established a correspondence between the A/B compartments in somatic interphase nucleus
and chromatin segments in giant lampbrush chromosomes from diplotene stage oocytes. The chromomere-loop
structure of the genomic regions corresponding to interphase A and B compartments reveals the difference in how
they are organized at the level of chromatin domains. The results obtained also suggest that gene-poor regions tend
to be packed into chromomeres. Keywords A/B compartments, Chicken genome, Chromomere, Chromosome conformation capture, FISH-mapping,
Hypertranscription, Lampbrush chromosomes, Meiotic chromosomes, Oocyte nucleus, Transcription loops © The Author(s) 2023. Background chromatin domains, the chromomeres, can be seen along
the whole length of the chromosome by conventional
light microscopy [29, 30]. Lateral loops emerging from
chromomeres are represented by strongly decondensed,
actively transcribed chromatin and are visible due to
the dense packing of nascent RNA with RNA-binding
proteins [31–33]. In birds, lampbrush chromomeres,
despite their primarily invariant pattern, are heterogene-
ous in terms of chromatin compactness, the presence and
length of lateral loops, epigenetic modifications, genomic
composition, and the abundance of repeats [30, 34–38]. FISH mapping of genomic loci belonging to somatic
chromatin domains has not enabled to establish an
unambiguous correlation between interphase TADs and
lampbrush chromomere-loop complexes [39]. g
Nowadays, the three-dimensional chromatin architecture
and the mechanisms of its maintenance and reorganiza-
tion is a rapidly developing area of genome biology [1]. With the advent of chromosome conformation cap-
ture approaches, the most widely used of which is the
genome-wide Hi-C method, chromatin domains such as
A/B compartments, topologically associating domains
(TADs), and loop domains have been described [2–5]. A
and B compartments are identified from the Hi-C long-
range contact frequency matrix by the “checkerboard”
pattern and represent large-scale chromatin domains
corresponding predominantly to open and closed chro-
matin [2, 6]. Chromatin visualization confirmed the
appearance of active and inactive nuclear compartments,
which form two interacting networks in the interphase
nucleus and generally match to A and B compartments
[7–9]. Moreover, A/B compartment domains may be
predicted from the histone modifications profile [10,
11]. A/B compartments have been detected in various
organisms, including human, Drosophila, plants, and
recently in the domestic chicken [3, 12–15]. A compart-
ments often correspond to transcriptionally active chro-
matin and enriched with epigenetic markers of an open
chromatin such as H3K36Me3, H3K4me1, H3K27ac,
H3K79me2 [3, 16, 17]. B compartments, on the other
hand, are usually gene-poor, compact, and contain his-
tone markers of a silent chromatin, such as H3K27me3,
H3K9Me3 [18]. In mammals, compartment domains can
be subdivided into subcomparments according to epi-
genetic and transcriptional profile [3, 19]. In contrast to
TADs, (sub-) compartment status and boundaries change
during cell differentiation that correlates with changes in
gene expression profiles [18, 20, 21]. At the same time,
there are (sub-) compartments with important house-
keeping functions that do not switch their status and
therefore can be called constitutive [20]. Background Lampbrush chromosomes are a convenient object for
chromatin mapping and have been studied in detail in a
number of model organisms [40–42]. However, the cor-
respondence between the chromomere-loop complexes
of meiotic lampbrush chromosomes and chromatin com-
partment identity in the interphase nucleus has not been
investigated. We decided to search for regularities in the
correspondence between chromatin domains in lamp-
brush chromosomes of the domestic chicken (Gallus gal-
lus domesticus) and A/B compartments in somatic cells
using available genome-wide data. To achieve this goal,
we set out to compare the pattern of lampbrush chro-
momeres with A/B compartment distribution along the
chromosomes and to map genomic regions belonging to
A or B compartments in chicken embryonic fibroblasts
on lampbrush chromosomes by FISH. To our knowl-
edge, this is the first study indicating a correspondence
between the A/B compartments in somatic interphase
nucleus and chromatin segments in giant lampbrush
chromosomes from diplotene stage oocytes. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 2 of 16 A/B compartment distribution analysis
d
fi d b Several studies indicate that in mammals, conven-
tional compartments are present in early oocytes but
become weaker during oocyte maturation and then dis-
appear in maternal zygotic chromatin [22, 23]. Here we
aimed to investigate how the compartmentalization of
the genome changes in growing oocytes in animals with
hypertranscriptional type of oogenesis. In this type of
oogenesis, high transcriptional output leads to appear-
ance of extremely elongated lampbrush chromosomes
with a typical chromomere-loop structure. Lampbrush
chromosomes are found in diplotene stage oocytes of
all vertebrates with the exception of mammals [24–28]. At this stage of oogenesis chromosomes nearly lack any
trans-chromosomal interactions except for the forma-
tion of bivalents between homologous chromosomes. In
each individual lampbrush chromosome, stable globular A/B compartments are identified by applying principal
component analysis (PCA or eigenvalue decomposition)
to the distance-normalized Hi-C interaction frequency
matrix for a certain cell type or tissue at a relatively low
resolution (usually, bin values are from several tens of
thousands kilobases to several megabases). The Pearson
correlation matrix is further used to calculate eigenvec-
tor for each matrix bin. The type of the compartment (A
or B) is generally inferred from the sign of the eigenvec-
tor: usually regions with positive values are denoted as
“A” compartments, while regions with negative values
are denoted as “B” compartments [2]. However, in some
cases, additional analysis of GC content or gene density
along the particular chromosome being analyzed may
be required to correct the eigenvector sign for better Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 3 of 16 alkaline-lysis protocol according to BAC manufacturer
instructions. BAC DNA was labeled by nick-translation
[58] at 16 °C for 2 h, using DNA polymerase I/DNAse I
enzyme mix (ThermoFisher Scientific) and either bio-
tin-11-dUTP (Lumiprobe), digoxigenin-11-dUTP (Jena
Bioscience) or aminoallyl-dUTP-ATTO-647N (Jena Bio-
science). Labeled probes were precipitated and dissolved
at 20–30 ng/μl in hybridization mixture, containing 50%
formamide, 10% dextran sulfate, 2 × SSC and 50 × excess
of salmon sperm DNA (ThermoFisher Scientific). Since
some BAC-clone based probes from the GGA1 gave
unspecific hybridization signal on other chromosomes
in metaphase plates, in case of these BAC clone combi-
nations, 20 × excess of chicken Cot5 DNA prepared by
S1-nuclease digestion was added to the hybridization
mixture [59]. The probes were preannealed at 37 °C for
1 h after denaturation before mounting on slides. prediction of the type of compartment [43]. Preparation of lampbrush chromosomes Lampbrush chromosomes were isolated from growing
chicken oocytes according to the earlier described pro-
cedure [60, 61] (https://projects.exeter.ac.uk/lampbrush/
protocols.htm) under stereomicroscope Leica S9D or
M165C (Leica Microsystems). Lampbrush chromosomes
were spread by 3000 rpm centrifugation for 30 min
at 4 °C, fixed in 2% PFA in PBS (1.47 mM KH2PO4,
4.29 mM Na2HPO4, 137 mM NaCl 2.68 mM KCl) for
30 min, dehydrated in ethanol series and air dried. Chick-
ens were handled according to the approval #131-04-6
dated 25.03.2019 of the Ethics Committee for Animal
Research of St. Petersburg State University. A/B compartment distribution analysis
d
fi d b It has been
convincingly shown that GC content and gene density
positively correlate with A-compartments and nega-
tively correlate with B compartments [44, 45]. A number
of computational program tools have now been devel-
oped to generate an A/B compartment profile from the
observed/expected Pearson correlation matrix, some of
which are embedded in pipelines for processing raw Hi-C
datasets, such as cooltools (https://open2c.github.io/) and
Juicer tools ([46]; https://github.com/aidenlab/juicer). In our study we used publicly available A/B compart-
ment profiles obtained for different chicken cell types:
chicken embryonic fibroblasts (CEF) and chicken eryth-
rocytes (RBC) [15], HD3 erythroblasts [47], lymphoblas-
toid DT40 cells [48], liver cells [49], and chicken small
white follicle (SWF) granulosa cells [50]. Since a galGal5
version of the chicken genome assembly was utilized to
generate Hi-C dataset for most cell types except SWF
cells, we used this chicken genome assembly to visualize
A/B compartment profiles in the Integrative Genomics
Viewer (IGV) [51]. Compartment coordinates for SWF
cells were remapped to GalGal5 genome assembly using
the NCBI remapping service (https://www.ncbi.nlm.nih.
gov/genome/tools/remap). Comparison of the lampbrush chromosome chromomeric
pattern with A/B compartment distribution A/B compartments in interphase nuclei of chicken
embryonic fibroblasts (CEF) were recently annotated in
Hi-C long-range chromatin interaction maps [15]. With
the help of the Integrative Genomics Viewer (IGV), we
visualized the distribution of A/B chromatin compart-
ments in CEF and other chicken cell types. In addi-
tion, we used previously developed cytological maps of
chicken lampbrush chromosomes with a DAPI staining
pattern [52–54] to compare the distribution of A/B com-
partments along GGA1–GGA7 chromosomes in somatic
cells with the distribution of chromomeres. To assign the
position of genomic regions, we used the coordinates of
previously mapped BAC clones [39, 53–56], the genomic
positions of centromeres and marker structures such as
globular loops [35, 57], and the coordinates of individual
chromomeres determined by microdissection and sub-
sequent sequencing [35, 37, 38]. The coordinates of the
selected markers were refined for the version 5 of the
domestic chicken genome (galGal5) (Additional file 1:
Table S1). FISH on lampbrush chromosomes DNA/DNA + RNA and DNA/RNA FISH protocols were
applied to lampbrush chromosome preparations [62,
63]. Lampbrush chromosomes were denatured in 70%
formamide at 70 °C for 10–15 min, dehydrated in ice-
cold ethanol series and air dried. Hybridization mixtures
were denatured at 95 °C for 10 min and transferred on
ice. 1–5 μl drops of hybridization mixture were applied
to lampbrush chromosome preparations covered with
coverslip and sealed with rubber cement. Hybridiza-
tion lasted overnight at 37 °C. Post-hybridization washes
were in 3 changes of 0.2 × SCC at 60 °C and 2 changes of
2 × 2CC at 45 °C. Biotin- and digoxigenin-labeled DNA-
probes were detected with Alexa488-conjugated strepta-
vidin and Cy3-conjugated anti-digoxigenin antibody
correspondingly. Labeling the BAC probes by nick‑translation
Probes were prepared from the chicken BAC-clone
library
CHORI-261
(https://bacpacresources.org/
chicken261.htm) (Additional file 1: Table S2). DNA
was extracted from E.coli night cultures by standard (See figure on next page.)
Fig. 1 Alignment of interphase genome A/B compartments along chicken chromosome 1 with chromomeric pattern of the corresponding
lampbrush chromosome. a—Distribution of A (red) and B (dark blue) compartments along the chicken chromosome 1 (GGA1) in embryonic
fibroblasts viewed by Integrative Genomics Viewer (IGV) (according to [15]); b—cytological map of chicken lampbrush chromosome 1 depicting
DAPI-staining pattern of chromomeres and relative contour length of lateral loops, black circles–dense chromomeres brightly stained with DAPI
(according to [53, 54]). Dotted lines in a, b connect the genomic positions of the selected BAC-clones (Additional file 1: Table S1) with their positions
on the cytological map; c—lampbrush chromosome 1 stained with DAPI, pixel intensities displayed with multicolored ImageJ look-up table,
numbered lines in a and c indicate positions of chromomere clusters brightly stained with DAPI; d, e, f—DNA+RNA-FISH with BAC-clone based
DNA-probes (Additional file 1: Table S2) covering the genomic regions 50–52 Mb (d), 70–71 Mb (e), and 185–186 Mb (f) on chicken lampbrush
chromosome 1; dotted lines from c to d–f indicate chromosomal positions of the regions on microphotographs; dʹ–fʹ—the positions of the
mapped BAC-clones relative to the somatic A/B compartments; dʹʹ–fʹʹ—schematic drawings of the FISH-mapping of the selected genomic regions
on lampbrush chromatin domains; colors correspond to the colors of the labeled DNA-probes on the FISH images. Scale bar: c–20 μm, d–f—10 μm Microscopy and image analysis Chromosomes were analyzed with epifluorescence
microscope Leica DM4000 (Leica Microsystems), images
were acquired with CCD camera (1.3 Mp resolution). Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 4 of 16 Krasikova et al. Epigenetics & Chromatin (2023) 16:24 DAPI-stained chromosomes were imaged before the
FISH procedure, and processed with ImageJ/Fiji mul-
ticolor look-up table ‘Fire’ to improve the visibility of
brightness heterogeneity along the lampbrush chro-
mosome axis. At least 6 images were acquired for each
chromosomal region to ensure accuracy and reproduci-
bility of FISH-mapping. Schematic drawings of the FISH-
mapping of the selected genomic regions on lampbrush
chromatin domains were generated according to the vis-
ual morphological analysis of the obtained microscopic
images. Each scheme represents a close approximation of
at least 6 different images with the mapped BAC probes
for each chromosomal region. The use of multiple FISH
images ensured that any variations in chromatin struc-
ture within the same chromosomal region were taken
into account and accurately represented in the final sche-
matic drawings. In addition, the giant size of the lamp-
brush chromosomes and their distinct chromomere-loop
structure defined a high level of detail in the schematic
drawings. DAPI-stained chromosomes were imaged before the
FISH procedure, and processed with ImageJ/Fiji mul-
ticolor look-up table ‘Fire’ to improve the visibility of
brightness heterogeneity along the lampbrush chro-
mosome axis. At least 6 images were acquired for each
chromosomal region to ensure accuracy and reproduci-
bility of FISH-mapping. Schematic drawings of the FISH-
mapping of the selected genomic regions on lampbrush
chromatin domains were generated according to the vis-
ual morphological analysis of the obtained microscopic
images. Each scheme represents a close approximation of
at least 6 different images with the mapped BAC probes
for each chromosomal region. The use of multiple FISH
images ensured that any variations in chromatin struc-
ture within the same chromosomal region were taken
into account and accurately represented in the final sche-
matic drawings. In addition, the giant size of the lamp-
brush chromosomes and their distinct chromomere-loop
structure defined a high level of detail in the schematic
drawings. clusters of prominent DAPI-positive chromomeres (clus-
ters # 1, 2, 4, 5, as well as not numbered smaller regions)
correspond to B compartments of different lengths in
chicken embryonic fibroblasts. Microscopy and image analysis i
Lampbrush chromosome GGA2 is characterized by the
presence of the so-called ‘‘spaghetti’’ marker on the short
arm and so-called lumpy loops (LLs) on the long arm
[61]. DAPI-positive chromomeres are evenly distributed
along the entire length of chromosome 2, with the large
clusters in the centromeric region and on the long arm. The locus of ‘‘spaghetti marker’’ formation, the coordi-
nates of which were established earlier [35], corresponds
to the A compartment, whereas the cluster of prominent
compact chromomeres, in which LLs form [65], corre-
sponds to the B compartment in embryonic fibroblasts
(Fig. 2a–c). The region of the extended cluster of dense
compact chromomeres (cluster #5), the boundaries of
which can be tentatively determined using previously
mapped BAC clones [53, 54], corresponds to the large B
compartment in embryonic fibroblasts (Fig. 2a–c).h i
The GGA3 lampbrush chromosome is well recognized
due to the characteristic asymmetric length of the lateral
loops, with a higher average loop length in the region of
the long arm closer to the centromere, giving noticeable
‘‘fuzziness’’ in the proximal part of the long arm, whereas
the distal part of the long arm is represented mainly by
DAPI-positive chromomeres with short lateral loops [52,
53]. These features are well reflected in the distribution
of epigenetic modifications: repressive chromatin mark-
ers H3K9me3 and 5-methyl-cytosine (5mC) predominate
in the clusters of chromomeres in the second half of the
long arm [38]. Extended clusters of large chromomeres in
this region of lampbrush chromosome 3 generally corre-
spond to the somatic B compartments (Additional file 1:
Figure S1a–aʹʹ). For instance, the region around the LL
marker of chromosome 3, which was mapped before [35],
demonstrated the correspondence between 5 and 6 dense
DAPI-positive chromomeres and homogenous B com-
partment in chicken embryonic fibroblasts. Lampbrush chromosome chromomeric pattern vs.
distribution of A/B chromatin compartments Lampbrush chromosome chromomeric pattern vs. distribution of A/B chromatin compartments First, we sought to compare the pattern of lampbrush
chromomeres with the distribution of A/B compart-
ments along the chicken interphase chromosomes, which
are characterized by multiple switches between A and B
compartments. The results of comparing the distribu-
tion of A/B compartments in chicken embryonic fibro-
blasts (CEF) with the pattern of chromomeres along the
lampbrush chromosomes GGA1–GGA7 are presented in
Figs. 1, 2, 3, 4 and Additional file 1: Figure S1. i
GGA1 lampbrush chromosome visually differs from
the other macrochromosomes by its long length, the
absence of any pronounced marker structures, with
the exception of the terminal loops (telomere bow-like
loops, TBLs) and one marker loop (proximal boundary
of axial bar bearing no loops, PBL11) [52, 53]. In lamp-
brush chromosome 1 several clusters of dense compact
chromomeres can be observed (Fig. 1a–c). Except for
the pericentromeric cluster of chromomeres, all other i
Lampbrush chromosome GGA4, in turn, has well-
defined terminal giant loops (TGLs) recently renamed
GITERA (Giant Terminal RNP aggregates) [66], two Page 5 of 16
Krasikova et al. Epigenetics & Chromatin (2023) 16:24
Fig. 1 (See legend on previous page.) Page 5 of 16
Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 5 of 16 Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 6 of 16 clusters of DAPI-positive chromomeres, one in the
short arm and one in the centromeric region, corre-
sponding to B compartments in embryonic fibroblasts
(Fig. 3a–c). Chicken lampbrush chromosome 4 is the
most completely mapped, both by FISH with BAC
clone-based probes [39, 53] and by microdissection of
individual chromomeres followed by their sequenc-
ing [37]. In the proximal part of the long arm of the
chromosome 4 is an extended cluster of small DAPI-
positive chromomeres (cluster #3) that are depleted in
acetylated histone H4 (H4Ac) and H3K9me3 [38]. This
region of GGA4 corresponds to intermingled A/B com-
partments in chicken embryonic fibroblasts with the
predominance of B compartment (Fig. 3a–c). In summary, by comparing the pattern of lampbrush
chromomeres with the distribution of A/B compart-
ments (Figs. Fig. 2 Alignment of interphase genome A/B compartments along chicken chromosome 2 with chromomeric pattern of the corresponding
lampbrush chromosome. a—Distribution of A (red) and B (dark blue) compartments along the chicken chromosome 2 (GGA2) in embryonic
fibroblasts viewed by Integrative Genomics Viewer (IGV) (according to [15]); b—cytological map of chicken lampbrush chromosome 2 depicting
DAPI-staining pattern of chromomeres and relative contour length of lateral loops, black circles—dense chromomeres brightly stained with
DAPI (according to [53, 54]). Dotted lines in a, b connect the genomic positions of the selected BAC-clones and chromosomal marker structures
(Additional file 1: Table S1) with their positions on the cytological map; c—lampbrush chromosome 2 stained with DAPI, pixel intensities displayed
with multicolored ImageJ look-up table, numbered lines in a and c indicate positions of chromomere clusters brightly stained with DAPI; d,
e—DNA+RNA-FISH with BAC-clone based DNA-probes (Additional file 1: Table S2) covering the genomic regions 39–40 Mb (d), 128–135 Mb (e)
on chicken lampbrush chromosome 2; dotted lines from c to d and e indicate chromosomal positions of the regions on microphotographs; dʹʹ,
eʹ—the positions of the mapped BAC-clones relative to the somatic A/B compartments; d, eʹʹ—schematic drawings of the FISH-mapping of the
selected genomic regions on lampbrush chromatin domains; colors correspond to the colors of the labeled DNA-probes on the FISH images. Scale
bar: c—20 μm, d, e—10 μm.
(See figure on next page.) FISH‑mapping of the genomic regions belonging to A or B
compartments on chicken lampbrush chromosomes Next, we decided to turn our attention to high resolution
mapping of the genomic regions belonging to A compart-
ments or regions transitional between A and B compart-
ments on lampbrush chromosome preparations. Based
on the available Hi-C data, we selected several regions of
interest to obtain probes for FISH mapping of A/B com-
partments present in chicken interphase genome. For the
most part (with a few exceptions), we focused on regions,
where the compartment sign was uniform at the 1 to
3 Mb length scales, on the largest macrochromosomes
(GGA1–4, GGA7) and one microchromosome (GGA14). Depending on the region, from 2 to 6 BAC clones from
the CHORI-261 library were selected to cover the A or
B compartment (Additional file 1: Table S2). The DNA
probes derived from the BAC clones to the selected
regions of interest were mapped on lampbrush chromo-
somes by 2D-FISH. FISH was performed according to the
DNA/DNA + RNA in situ hybridization protocol so that
DNA-probes also hybridized with the nascent transcripts
on the lateral loops, allowing the hybridization signal to
be observed along the entire length of the transcription
loop. GGA5 lampbrush chromosome is characterized
by several clusters of larger and more globular chro-
momeres including a cluster without prominent lateral
loops in the pericentromeric region [56]. Four of these
clusters correlate with the B compartments present at
chicken interphase genome (Additional file 1: Figure S1
b–bʹʹ). GGA6 in a lampbrush configuration can be divided
into two parts—with more compact larger chromomeres
and relatively short lateral loops and with tiny chro-
momeres and longer transcription loops [53]. Prominent
B compartment can be assigned to the first part of lamp-
brush chromosome 6 with higher chromatin compaction
(cluster #1) (Additional file 1: Figure S1c–cʹʹ).h i
The GGA7 midichromosome, like the other midichro-
mosomes, is characterized by small DAPI-negative chro-
momeres from which long lateral loops emerge. Based on
the analysis of DAPI staining and counting of the aver-
age number of chromomeres, we drew an approximate
cytological map of the chromomere pattern for GGA7
(Fig. 4a–c). Clusters of DAPI-positive chromomeres are
found in the centromeric region, the position of which
was previously established [57], and in the distal region
of the long arm. B compartments are detected in the
genomic region corresponding to these clusters, but
further FISH mapping is required to verify their corre-
spondence to the DAPI-positive chromomeres. Lampbrush chromosome chromomeric pattern vs.
distribution of A/B chromatin compartments 1, 2, 3, 4a–c, Additional file 1: Figure S1), we
have established that lampbrush chromosome segments
characterized by higher chromatin compaction, more
globular chromomeres, and lower transcriptional activity
generally correlate with the B compartments present at
interphase chromatin. At the same time, no obvious cor-
respondence with either A or B compartment identity is
found for smaller chromomeres with relatively long lat-
eral loops. (See figure on next page.)
Fig. 3 Alignment of interphase genome A/B compartments along chicken chromosome 4 with chromomeric pattern of the corresponding
lampbrush chromosome. a—Distribution of A (red) and B (dark blue) compartments along the chicken chromosome 4 (GGA4) in embryonic
fibroblasts viewed by Integrative Genomics Viewer (IGV) (according to [15]); b—cytological map of chicken lampbrush chromosome 4 depicting
DAPI-staining pattern of chromomeres and relative contour length of lateral loops, black circles—dense chromomeres brightly stained with DAPI
(according to [53]). Dotted lines in a, b connect the genomic positions of the selected BAC-clones (Additional file 1: Table S1) with their positions on
the cytological map; c—lampbrush chromosome 4 stained with DAPI, pixel intensities displayed with multicolored ImageJ look-up table, numbered
lines in a and c indicate positions of chromomere clusters brightly stained with DAPI; d—DNA+RNA-FISH with BAC-clone based DNA-probes
(Additional file 1: Table S2) covering the genomic region 34–37 Mb (d) on chicken lampbrush chromosome 4; dotted lines from c to d indicate
chromosomal position of the region on microphotographs; dʹ—the positions of the mapped BAC-clones relative to the somatic A/B compartments;
dʹ—schematic drawing of the FISH-mapping of the selected genomic region on lampbrush chromatin domains; colors correspond to the colors of
the labeled DNA-probes on the FISH image. Scale bar: c—20 μm, d—10 μm. FISH‑mapping of the genomic regions belonging to A or B
compartments on chicken lampbrush chromosomes Mapping genomic regions belonging to A compartments
To map the genomic regions belonging to A compart-
ments we selected the following segments on four chro-
mosomes: 50–52 Mb in GGA1, 39–40 Mb in GGA2, Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 7 of 16 Krasikova et al. Epigenetics & Chromatin Page 7 of 1
rasikova et al. Epigenetics & Chromatin (2023) 16:24
Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 8 of 16 12–14 Mb in GGA7, and 1–2 Mb in GGA14 (Additional
file 1: Table S2). chromosomes isolated from the oocytes. To this end,
70–71 Mb region in GGA1, 128–135 Mb region in
GGA2, 34–37 Mb region in GGA4, and 10–13 Mb
region in GGA14 were mapped (Additional file 1:
Table S2). i
Six BAC clones were selected for the 50–52 Mb region
of chromosome 1 (Fig. 1dʹ). FISH mapping of the selected
BAC clones on lampbrush chromosome 1 shows the
presence of chromomere-loop complexes with long lat-
eral loops in the region corresponding to A compartment
(Fig. 1d–dʹʹ). The 70–71 Mb region of GGA1 is at the border
between the chromosomal segment with very long lat-
eral loops and the cluster of centromeric DAPI-positive
chromomeres (the so-called ‘centromere bar’). This seg-
ment of the lampbrush chromosome 1 corresponds to
the interphase chromatin region with multiple switches
between A and B compartments (Fig. 1a–c). BAC clones
CH261-162E14,
CH261-33I15,
and
CH261-180H2
from this region hybridized with two neighboring chro-
momeres with pairs of short lateral loops (Fig. 1e–eʹʹ). BAC
clones
CH261-163C1,
CH261-63A12,
and
CH261-134B20 were selected to the 39–40 Mb region
of chicken chromosome 2 (GGA2) (Fig. 2dʹ). All three
probes hybridized with the RNP-matrix of the lateral
loops (Fig. 2d–d′′). BAC
clones
CH261-163C1,
CH261-63A12,
and
CH261-134B20 were selected to the 39–40 Mb region
of chicken chromosome 2 (GGA2) (Fig. 2dʹ). All three
probes hybridized with the RNP-matrix of the lateral
loops (Fig. 2d–d′′). Three BAC clones CH261-93F1, CH261-126G14, and
CH261-38J23 selected to the 12–14 Mb region of chicken
chromosome 7 (GGA7) were mapped into a single pair
of long lateral loops with two transcriptional units sepa-
rated by a chromatin nodule (Fig. 4d–dʹʹ). FISH‑mapping of the genomic regions belonging to A or B
compartments on chicken lampbrush chromosomes The presence
of a chromatin nodule within the lateral loop can be
explained by the fact that in the absence of transcribed
genes, chromatin tends to persist in a compact, ‘‘closed’’
state, even without being anchored into a nearby chro-
momere. Chromatin nodules on the lateral loops of
lampbrush chromosomes have been described previously
at the morphological level as 5mC enriched transcript-
free DNP regions with the characteristic nucleosomal
organization [67] and by BAC-clone mapping as untran-
scribed DNA regions [39]. We also selected two differently labeled BAC clone
sets to map an extended 128–135 Mb genomic seg-
ment of transition from the A to the B compartment on
chicken chromosome 2 (GGA2) (Fig. 2eʹ). BAC clones
CH261-98G7, CH261-135E13 picked up to the A com-
partment hybridized into chromomere-loop complexes
with long lateral loops, indicating chromatin openness
in this region. Next, BAC clones CH261-12O18, CH261-
140E6, CH261-177C13, CH261-97C18, and CH261-54I9,
matched to the B compartment, hybridized into chro-
momeres (Fig. 2e–eʹʹ). One of these BAC clones also
hybridized with a very short lateral loop, which may be
related to the transcription of a certain gene. This hybrid-
ization pattern fully confirms our observation about
the preferential correspondence of A compartments to
smaller chromomeres with longer lateral loops and B
compartments—to larger chromomeres with shorter lat-
eral loops. Two BAC clones, CH261-94D13, CH261-168C19 to a
1–2 Mb region of chicken chromosome 14 (GGA14) also
belonging to A compartment hybridized with the RNP
matrix of two long lateral loops (Fig. 5c–cʹʹ). Overall, all four genomic regions belonging to the A
compartments in somatic cells were mapped to the rela-
tively long lateral loops and adjacent rather small chro-
momeres in lampbrush chromosome chromatin. Two other transition genomic regions: the 34–37 Mb
segment on chromosome 4 and the 10–13 Mb seg-
ment on chromosome 14 demonstrated an exception to
the observed tendency. Four BAC clone-based probes,
CH261-30O11, CH261-38A10 to the A compartment
and CH261-124F10, CH261-109C8 to the B compart-
ment, were selected for the 34–37 Mb region on chromo-
some 4 (GGA4) (Fig. 3dʹ). All four probes hybridized into
the RNP-matrix of four adjacent lateral loops of different Mapping genomic regions transitional between A and B
compartments We additionally selected BAC clones for A to B
compartment transition regions in chicken embry-
onic fibroblasts for FISH-mapping on lampbrush Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 9 of 16 Page 9 of 16
a et al. Epigenetics & Chromatin (2023) 16:24 3 (See legend on previous page.) Fig. 3 (See legend on previous page.) Fig. 3 (See legend on previous page.) Fig. 3 (See legend on previous page.) Fig. 3 (See legend on previous page.) Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 10 of 16 Page 10 of 16 sizes regardless of whether they belonged to the A or B
compartment (Fig. 3d–dʹʹ). hybridized with a pair of very short lateral loops, ema-
nating from the central chromomere (Fig. 1f–fʹʹ). Thus,
this cluster of DAPI-positive chromomeres on lamp-
brush chromosome 1 (cluster #5) (Fig. 1c) corresponds
to the genomic region dominated by B compartments
(Fig. 1a–b). Tiny lateral loops that emerge from these
chromomeres are aligned with the small A compartment
within this genomic region (Fig. 1f–fʹʹ). This example
confirms the general correspondence between genomic
regions forming B compartment in chicken embry-
onic fibroblasts and the large clusters of dense chro-
momeres with short lateral loops in lampbrush meiotic
chromosomes. The GGA14 microchromosome contains two clusters
of CNM repeats in the centromeric region corresponding
to two large DAPI-positive chromomeres at the lamp-
brush chromosome stage [54] and an additional extended
B compartment in the middle of the proximal part of the
long arm (Fig. 5a–b). At the same time, three BAC clones
from this B compartment mapped to three pairs of lat-
eral loops, indicating active transcription of this region at
the lampbrush chromosome stage [39]. In particular, the
748 kbp gene RBFOX1 (RNA Binding Fox-1 Homolog 1)
present in this region is transcribed on a very long lateral
loop [39] but is silent in chicken embryonic fibroblasts. In addition, the BAC probes that we earlier mapped
on chicken lampbrush chromosome 1 to genomic loci
from the two neighboring TADs belonging to the com-
partment B (146–148 Mb region) hybridize with one
prominent chromomere, but not with lateral loops
(Fig. 2 in [39]). Previously, microdissection and sequenc-
ing of a large dense chromomere from this region (149–
154 Mb) showed that it is enriched with H3K9me3 and
highly methylated gene-poor DNA corresponding to
the somatic B compartment [35, 38]. Fig. 4 Alignment of interphase genome A/B compartments along chicken chromosome 7 with chromomeric pattern of the corresponding
lampbrush chromosome. a—Distribution of A (red) and B (dark blue) compartments along the chicken chromosome 7 (GGA7) in embryonic
fibroblasts viewed by Integrative Genomics Viewer (IGV) (according to [15]); b—cytological map of chicken lampbrush chromosome 7 depicting
DAPI-staining pattern of chromomeres and relative contour length of lateral loops, black circles–dense chromomeres brightly stained with DAPI.
Dotted lines in a, b connect the genomic positions of the BAC-clones (Additional file 1: Table S1) with their positions on the cytological map; c—
lampbrush chromosome 7 stained with DAPI, pixel intensities displayed with multicolored ImageJ look-up table, numbered line in a and c indicates
position of chromomere cluster brightly stained with DAPI; d, dʹ–DNA+RNA-FISH with BAC-clone based DNA-probes (Additional file 1: Table S2)
covering the genomic region 12–14 Mb on chicken lampbrush chromosome 7; dotted lines from c to d, d′ indicate chromosomal positions of
the region on microphotographs; dʹʹ—schematic drawing of the FISH-mapping of the selected genomic region (d, dʹ) on lampbrush chromatin
domains; colors correspond to the colors of the labeled DNA-probes on the FISH images. e—the positions of the mapped BAC-clones relative to
the somatic A/B compartments. Scale bar: c—20 μm, d, dʹ—10 μm.
(See figure on next page.) Mapping genomic regions transitional between A and B
compartments In addition, among
three BAC clones to the three subTADs from the com-
partment B (25–28 Mb region) on lampbrush chro-
mosome 2 (Fig. 2a–c, region 2), two hybridized with
chromomeres and one hybridized with the nascent RNA
on a pair of lateral loops corresponding to the TSHD7A
gene (Fig. 3 in [39]). p
yi
Here, we selected 9 additional and one previously
mapped BAC clones for this region of GGA14–6 to the
B compartment (CH261-177N7, CH261-36G10, CH261-
179I1, CH261-119E18, CH261-78O7, CH261-99K17)
and 4 to the A compartment (CH261-75C12, CH261-
75E9, CH261-57E13, CH261-152F2) (10–13 Mb region,
Fig. 5dʹ). Probes to the B compartment hybridized
with the RNP matrix of two long adjacent lateral loops
(Fig. 5d–dʹʹ). Probes to the A compartment also hybrid-
ized with the lateral loops, but despite the abundance of
the genes in this region, the size of these loops was much
smaller. This may be related both to the different lengths
of the transcribed genes and to the intensity of their tran-
scription at this stage of oogenesis. We assume that in
the 10–13 Mb region of GGA14, the longest lateral loops
are formed in the genomic regions containing long tran-
scribed genes regardless of the compartment status. In conclusion, our FISH-mapping data additionally
indicate that clusters of dense compact chromomeres
carrying short lateral loops and enriched with repres-
sive epigenetic modifications overlap with prominent
B compartments in somatic cells, reflecting functional
compartmentalization of the chicken genome. Next,
we wondered if these compartments are constitutive or
cell-type specific. Using the data available up to date, we
demonstrate that these B compartments are constitutive
between several chicken cell types: embryonic fibroblasts
(CEF), mature erythrocytes (RBC), erythroblast cell line
(HD3), DT40 cell line, granulosa cells of small white fol-
licle (SWF), and liver cells (Additional file 1: Figure S2). Mapping genomic regions belonging to B compartments Mapping genomic regions belonging to B compartments
To map the genomic regions belonging to the somatic
B compartments, we chose a 185–186 Mb genomic seg-
ment that belongs to GGA1 region, dominated by B com-
partments interspersed with smaller A compartments
(cluster #5) (Fig. 1a), and selected three neighboring
BAC clones CH261-31K17, CH261-54J7, CH261-120O20
(Fig. 1fʹ). BAC clone-based probes CH261-31K17,
CH261-54J7, and CH261-120O20 hybridized to three
adjacent chromomeres and probe CH261-54J7 also Fig. 4 Alignment of interphase genome A/B compartments along chicken chromosome 7 with chromomeric pattern of the corresponding
lampbrush chromosome. a—Distribution of A (red) and B (dark blue) compartments along the chicken chromosome 7 (GGA7) in embryonic
fibroblasts viewed by Integrative Genomics Viewer (IGV) (according to [15]); b—cytological map of chicken lampbrush chromosome 7 depicting
DAPI-staining pattern of chromomeres and relative contour length of lateral loops, black circles–dense chromomeres brightly stained with DAPI. Dotted lines in a, b connect the genomic positions of the BAC-clones (Additional file 1: Table S1) with their positions on the cytological map; c—
lampbrush chromosome 7 stained with DAPI, pixel intensities displayed with multicolored ImageJ look-up table, numbered line in a and c indicates
position of chromomere cluster brightly stained with DAPI; d, dʹ–DNA+RNA-FISH with BAC-clone based DNA-probes (Additional file 1: Table S2)
covering the genomic region 12–14 Mb on chicken lampbrush chromosome 7; dotted lines from c to d, d′ indicate chromosomal positions of
the region on microphotographs; dʹʹ—schematic drawing of the FISH-mapping of the selected genomic region (d, dʹ) on lampbrush chromatin
domains; colors correspond to the colors of the labeled DNA-probes on the FISH images. e—the positions of the mapped BAC-clones relative to
the somatic A/B compartments. Scale bar: c—20 μm, d, dʹ—10 μm. (See figure on next page.) Page 11
et al. Epigenetics & Chromatin (2023) 16:24 Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Krasikova et al. Epigenetics & Chromatin Page 11 of 16 sion
uestion of whether the structure of lampbrush
osomes is unique or reflects universal principles
of genome packaging remains open. In transcr
ally active lampbrush chromosomes, probably
the strong repulsion of RNP-matrix of lateral
(See legend on previous page.) . 4 (See legend on previous page.) Fig. 4 (See legend on previous page.) Fig. 4 (See legend on previous page.) of genome packaging remains open. In transcription-
ally active lampbrush chromosomes, probably due to
the strong repulsion of RNP-matrix of lateral loops, (See figure on next page.)
Fig. 5 Alignment of interphase genome A/B compartments along chicken chromosome 14 with chromomeric pattern of the corresponding
lampbrush chromosome. a—Distribution of A (red) and B (dark blue) compartments along the chicken chromosome 14 (GGA14) in embryonic
fibroblasts viewed by Integrative Genomics Viewer (IGV) (according to [15]); b—cytological map of chicken lampbrush chromosome 14 depicting
DAPI-staining pattern of chromomeres and relative contour length of lateral loops, black circles—dense chromomeres brightly stained with DAPI
(according to [54]). Dotted lines in a, b connect the genomic positions of the BAC-clones (Additional file 1: Table S1) with their positions on the
cytological map; c, d—DNA + RNA-FISH with BAC-clone based DNA-probes (Additional file 1: Table S2) covering the genomic regions 1–2 Mb cʹ
and 10–13 Mb dʹ on chicken lampbrush chromosome 14, dotted lines from b to c and d indicate positions of the regions on the cytological map;
cʹ, dʹ—the positions of the mapped BAC-clones relative to the somatic A/B compartments; cʹʹ, dʹʹ—schematic drawing of the FISH-mapping of the
selected genomic regions (c, d) on lampbrush chromatin domains; colors correspond to the colors of the labeled DNA-probes on the FISH images.
Scale bar: 10 μm Discussionh The question of whether the structure of lampbrush
chromosomes is unique or reflects universal principles Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 12 of 16 Page 12 of 16 suggest that gene-poor regions tend to be packed in chro-
momeres in lampbrush chromosomes. The boundaries of
extended clusters of compact chromomeres correspond-
ing to particular B compartments were established pre-
cisely according to the mapped genetic markers. Mapping
of the transition regions between A and B compartments
further supports the observed tendency. We also assume
that in the most genomic regions analyzed, A compart-
ments in chicken embryonic fibroblasts tend to corre-
spond to the lampbrush chromosome segments with
smaller and less compact chromomeres and long lateral
loops, which correlates with their higher transcriptional
status, enrichment with histone H4 acetylation, and elon-
gating form of RNA polymerase II. These chromosomal
segments have a greater number of less globular chro-
momeres compared to the segments overlapping with
the somatic B compartments. At the same time, smaller
chromomeres with relatively long lateral loops show no
obvious correspondence with either A or B compartment
identity. A more detailed examination of the distribution
of different types of subcompartments and functional
classes of chromatin in these genomic regions and their
relationship to particular lampbrush chromatin domains
is essential. chromatin compartments are apparently absent. Indeed,
in somatic cells, genomic loci from one particular B com-
partment preferentially interact with genomic loci form
the other B compartments. However, in giant oocyte
nucleus, individual chromosomal segments do not
reach each other, both between and within lampbrush
chromosomes [68, 69]. Moreover, in growing oocytes
of adult birds, lampbrush chromosomes do not interact
with nuclear lamina or nucleolus and do not form chro-
mocenters, which reduces the likelihood of interchromo-
somal interactions. Drosophila polytene chromosomes
lack compartments due to similar spatial organization
[70]. Previously, based on Hi-C chromatin contact maps, the
distribution of TADs and A/B compartments in inter-
phase genome was compared with G-banding (according
to Giemsa staining) of human metaphase chromosomes
[71]. It was found that TADs with high H1.2/H1X ratio
belonging to B compartments strongly correlate with
AT-rich Giemsa bands. Our data further indicate that
the boundaries between chromatin domains belonging
to A/B compartments in somatic cells remain bounda-
ries and in lampbrush chromosomes from diplotene
stage oocytes. Discussionh At the same time, genomic regions corre-
sponding to the extended chromatin domains restricted
by compartment boundaries in somatic cells disintegrate
into individual chromomeres in diplotene oocytes. We also realized that there are a number of exceptions
to the observed trend. For example, we noticed that tran-
scription of the lengthy RBFOX1 gene during oogenesis
leads to the formation of a long lateral loop in the region
corresponding to the B compartment in somatic cells
(Fig. 5d–dʹʹ). This is interconnected to oocyte specific
expression of many maternal genes as shown by RNA-
FISH on lampbrush chromosome preparations [39, 63]. Thus, this B-compartment can be regarded as facultative. The schematic drawing (Fig. 6) summarizes the estab-
lished correspondence between chromatin domains
belonging to A or B compartments in chicken interphase
genome and lampbrush chromosome segments with dif-
ferent chromomere-loop characteristics. The following
issues may be addressed in the future. What is the mech-
anism of chromatin segregation into individual chro-
momeres in lampbrush meiotic chromosomes? Whether
chromomeres represent chromatin that is passively
compacted due to transcriptional inactivity or whether We also realized that there are a number of exceptions
to the observed trend. For example, we noticed that tran-
scription of the lengthy RBFOX1 gene during oogenesis
leads to the formation of a long lateral loop in the region
corresponding to the B compartment in somatic cells
(Fig. 5d–dʹʹ). This is interconnected to oocyte specific
expression of many maternal genes as shown by RNA-
FISH on lampbrush chromosome preparations [39, 63]. Thus, this B-compartment can be regarded as facultative. y
In fact, chicken lampbrush chromosomes are charac-
terized by distinct heterogeneity in chromomere density
and contour length of lateral loops. Clusters of dense
and relatively large chromomeres with short lateral loops
alternate with regions of loose and sometimes indiscern-
ible chromomeres with longer lateral loops [52, 53, 72]. It was found that the repressive chromatin modifica-
tions (5mC, H3K9me3, H3K27me3, HP1β) are enriched
in the clusters of dense compact chromomeres reflect-
ing their functional state [36, 38]. By direct comparison
of Hi-C maps and chromomere-loop patterns as well
as by FISH-mapping, we found that clusters of more
globular chromomeres with relatively short lateral loops
and thus higher chromatin compaction generally cor-
respond to the constitutive B compartments present in
the interphase nucleus of many cell types. These results h
The schematic drawing (Fig. Discussionh 6) summarizes the estab-
lished correspondence between chromatin domains
belonging to A or B compartments in chicken interphase
genome and lampbrush chromosome segments with dif-
ferent chromomere-loop characteristics. The following
issues may be addressed in the future. What is the mech-
anism of chromatin segregation into individual chro-
momeres in lampbrush meiotic chromosomes? Whether
chromomeres represent chromatin that is passively
compacted due to transcriptional inactivity or whether Page 13
Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 13 of 16 Fig. 5 (See legend on previous page.) Fig. 5 (See legend on previous page.) Fig. 5 (See legend on previous page.) Fig. 5 (See legend on previous page.) Krasikova et al. Epigenetics & Chromatin (2023) 16:24 Page 14 of 16 A-compartment
Lampbrush
chromatin
domains
B-compartment genomic regions with higher gene density are actively
transcribed at the lampbrush chromosome stage of
oogenesis. B-compartment Funding Funding
The study was supported by the RSF grant #19-74-20075. Availability of data and materials Availability of data and materials
All data are included in the manuscript files. All data are included in the manuscript files Acknowledgements
f Fig. 6 Schematic drawing generalizing the correspondence between
A or B compartments present in interphase genome and lampbrush
chromosome segments. Chromosomal segments with more globular
compact chromomeres and short lateral loops correspond to B
compartment (blue), whereas chromosomal segments composed by
small loose chromomeres with long transcription loops correspond
to A compartment (red) Microscopy was performed using the equipment of the Resource Center
“Molecular and Cell Technologies” (Research Park of Saint-Petersburg State
University). Ethics approval and consent to participate Chickens were handled according to the approval #131-04-6 dated
25.03.2019 of the Ethics Committee for Animal Research of St. Petersburg
State University. Author contributions AK conceptualized the study and supervised the project. JS RR, TK and AK
compared the distribution of compartments with lampbrush chromomere
pattern. AM and AK selected regions of interest and BAC clone-based probes,
AM labeled probes by nick-translation. TK and JS RR performed FISH on iso-
lated lampbrush chromosomes and analyzed the micrographs. AK suggested
the final scheme. AK and TK wrote the manuscript with a contribution from
co-authors. All authors read and approved the final manuscript. chromatin in chromomeres is protected from transcrip-
tion initiation due to a more closed chromatin struc-
ture? It would be also interesting to compare lampbrush
chromomeres with chromatin domains and chromatin
domain clusters forming chain-like reticular structure
recently described in interphase nucleus by quantitative
super-resolution and scanning electron microscopy [7,
73]. The online version contains supplementary material available at https://doi.
org/10.1186/s13072-023-00499-2. The online version contains supplementary material available at https://doi.
org/10.1186/s13072-023-00499-2. Lampbrush
chromatin
domains Additional file 1: Figure S1. Distribution of A/B compartments along the
chicken chromosomes 3, 5 and 6 in embryonic fibroblasts compared with
the chromomeric pattern in the corresponding lampbrush chromosomes. Figure S2. Examples of constitutive B compartment regions in previ-
ously studied chicken cell types. Table S1. Genomic regions mapped on
chicken lampbrush chromosomes and marked on the coordinate line on
Figures 1, 2, 3, 4, 5, Additional file 1: Figure S1, according to the chicken
genome version 5 (galGal5). Table S2. The list of BAC clones containing
fragments of chicken genomic DNA from the CHORI-261 library that were
used as DNA-probes for FISH; coordinates are indicated according to the
chicken genome version 5 (galGal5). Cluster of large and
dense chromomeres
with short lateral loops Cluster of small and
loose chromomeres
with long lateral loops Chromatin:
- open
- decondensed
- transcriptionally active
- RNA-pol II enriched Chromatin: Chromatin:
- closed
- condensed
- large portion is
transcriptionally silent - closed - closed - condensed - condensed
- large portion is
transcriptionally silent - large portion is
transcriptionally silent Author details
1 Author details
1 Saint-Petersburg State University, Saint‑Petersburg, Russia. 1 Saint-Petersburg State University, Saint‑Petersburg, Russia. Received: 7 April 2023 Accepted: 4 June 2023 Received: 7 April 2023 Accepted: 4 June 2023 Competing interests Competing interests
None of the authors have any competing interests. p
g
None of the authors have any competing interests. g
None of the authors have any competing interests. Conclusions We compared the distribution of A/B compartments in
somatic cells with chromatin domains in meiotic lamp-
brush chromosomes. The chromomere-loop structure of
the genomic regions corresponding to interphase A and
B compartments reveals the difference in how they are
organized at the level of chromatin domains. In conclu-
sion, clusters of globular chromomeres with shorter lat-
eral loops, resulting in higher chromatin compaction, are
commonly associated with constitutive B compartments
in interphase nuclei of different cell types. This obser-
vation suggests that genomic regions with lower gene
density tend to be concentrated within chromomeres in
lampbrush chromosomes. We further inferred that A
compartments in chicken embryonic fibroblasts align
with lampbrush chromosome segments with smaller,
less compact chromomeres, longer lateral loops, and a
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? Choose BMC and benefit from: 72. Solovei I, Gaginskaya E, Hutchison N, Macgregor H. Avian sex chromo-
somes in the lampbrush form: the ZW lampbrush bivalents from six
species of bird. Chromosome Res. 1993;1:153–66. 73. Miron E, Oldenkamp R, Brown JM, Pinto DMS, Xu CS, Faria AR, et al. Chromatin arranges in chains of mesoscale domains with nanoscale
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70. Eagen KP, Hartl TA, Kornberg RD. Stable chromosome condensation
revealed by chromosome conformation capture. Cell. 2015;163:934–46. 71. Serna-Pujol N, Salinas-Pena M, Mugianesi F, Lopez-Anguita N, Torrent-
Llagostera F, Izquierdo-Bouldstridge A, et al. TADs enriched in histone
H1.2 strongly overlap with the B compartment, inaccessible chromatin,
and AT-rich Giemsa bands. FEBS J. 2021;288:1989–2013. 71. Serna-Pujol N, Salinas-Pena M, Mugianesi F, Lopez-Anguita N, Torrent-
Llagostera F, Izquierdo-Bouldstridge A, et al. TADs enriched in histone
H1.2 strongly overlap with the B compartment, inaccessible chromatin,
and AT-rich Giemsa bands. FEBS J. 2021;288:1989–2013. •
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Symbitron Exoskeleton: Design, Control, and Evaluation of a Modular Exoskeleton for Incomplete and Complete Spinal Cord Injured Individuals
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IEEE TRANSACTIONS ON NEURAL SYSTEMS AND REHABILITATION ENGINEERING, VOL. 29, 2021 330 N. L. Tagliamonte, F. Tamburella, I. Pisotta, M. Masciullo, M. Arquilla,
and M. Molinari are with the NeuroRobot Lab, Fondazione Santa Lucia,
00179 Rome, Italy. j
)
G. van Oort, V. Sluiter, and E. van Asseldonk are with the Department
of Biomechanical Engineering, University of Twente, 7522 NB Enschede,
The Netherlands. Symbitron Exoskeleton: Design, Control, and
Evaluation of a Modular Exoskeleton for
Incomplete and Complete Spinal
Cord Injured Individuals C. Meijneke
, G. van Oort
, V. Sluiter, E. van Asseldonk
, N. L. Tagliamonte, F. Tamburella
,
I. Pisotta
, M. Masciullo
, M. Arquilla, M. Molinari
, A. R. Wu
, F. Dzeladini,
A. J. Ijspeert
, Fellow, IEEE, and H. van der Kooij could not walk without support, were able to walk with the
device and with the support of crutches that included a
push-button for step initiationOur results demonstrate that
an exoskeleton with modular hardware and control allows
SCI individuals with limited or no lower limb function to
receive tailored support and regain mobility. Abstract— In this paper, we present the design, control,
and preliminary evaluation of the Symbitron exoskeleton,
a lower limb modular exoskeleton developed for people
with a spinal cord injury. The mechanical and electrical
configuration and the controller can be personalized to
accommodate differences in impairments among individ-
uals with spinal cord injuries (SCI). In hardware, this per-
sonalization is accomplished by a modular approach that
allows the reconfiguration of a lower-limb exoskeleton with
ultimately eight powered series actuated (SEA) joints and
high fidelity torque control. For SCI individuals with an
incomplete lesion and sufficient hip control, we applied a
trajectory-free neuromuscular control (NMC) strategy and
used the exoskeleton in the ankle-knee configuration. For
complete SCI individuals, we used a combination of a NMC
and an impedance based trajectory tracking strategy with
the exoskeletonin the ankle-knee-hipconfiguration.Results
of a preliminary evaluation of the developed hardware and
software showed that SCI individuals with an incomplete
lesion could naturally vary their walking speed and step
length and walked faster
compared to walking without
the device. SCI individuals with a complete lesion, who Index
Terms— Exoskeleton,
modular,
orthosis,
SCI,
series elastic actuation (SEA), neuromuscular control
(NMC). j
(
p
g
j
)
C. Meijneke is with the Electronic and Mechanical Support Division,
Delft University of Technology, 2628 CD Delft, The Netherlands (e-mail:
c.meijneke@tudelft.nl). A. R. Wu, F. Dzeladini, and A. J. Ijspeert are with the Biorobot-
ics (BIOROB) Laboratory, École Polytechnique Fédérale de Lausanne,
1015 Lausanne, Switzerland. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION I I
NDIVIDUALS with spinal cord injuries (SCI) experience
reduced or complete loss of mobility, but wearable tech-
nologies such as lower-limb exoskeletons could be used to
assist gait and help improve their quality of life. Several
commercially available exoskeletons have been developed for
and used by subjects with a complete SCI [1]. They mostly
consist of a structure that allows actuation of knee and hip
flexion and extension movements. The exoskeleton joints are
typically controlled in a position control mode, where the
motions of the human joints are enforced and cannot be
influenced by the user. However, about 60% of individuals
who have suffered a spinal cord injury only have an incom-
plete lesion and have some remaining function in their lower
extremities [2]. The remaining movement capabilities depend
on the severity and location of the lesion; the lower (the more
inferior/caudal) the lesion, the more remaining function in the
lower leg joints starting from the most proximal joints (the
hip) to more distal joints [3]. Exoskeletons also have great
potential to improve the walking ability of individuals with a
incomplete lesion (in terms of speed, endurance and stability)
if they can substitute for the lost or impaired function and
leave control of the unaffected joints to the individual. This
approach requires a different class of exoskeletons that can be
tailored to the individual’s needs and provides assisting forces
without enforcing movements. Manuscript received July 30, 2020; revised December 10, 2020;
accepted January 5, 2021. Date of publication January 8, 2021; date
of current version March 2, 2021. This work was supported in part
by the SYMBITRON Project through the EU Research Program FP7,
FET-Proactive Initiative (Symbiotic human-machine interaction) (ICT-
2013-10) under Project 611626, and in part by the Nederlandse Organ-
isatie voor Wetenschappelijk Onderzoek [Netherlands Organization for
Scientific Research (NWO)] Domain Applied and Engineering Sciences
under Project 14429. (Corresponding author: C. Meijneke.) G. van Oort, V. Sluiter, and E. van Asseldonk are with the Department
of Biomechanical Engineering, University of Twente, 7522 NB Enschede,
The Netherlands. N. L. Tagliamonte, F. Tamburella, I. Pisotta, M. Masciullo, M. Arquilla,
and M. Molinari are with the NeuroRobot Lab, Fondazione Santa Lucia,
00179 Rome, Italy. A. R. Wu, F. Dzeladini, and A. J. Ijspeert are with the Biorobot-
ics (BIOROB) Laboratory, École Polytechnique Fédérale de Lausanne,
1015 Lausanne, Switzerland. H. I. INTRODUCTION Previously we have incorporated these models as controllers
(called neuromuscular controllers, NMC) for a powered ankle
exoskeleton [6]. In a follow up study, we demonstrated with
this wearable Achilles ankle exoskeleton that incomplete SCI
individuals improved their walking speed and step length
wearing the device over different training sessions, and we
also found an unexpected increased walking speed using no
device after a brief training period with the exoskeleton [7]. NMC controllers for the knee and hips were also rendered
on the haptic robotic gait trainer LOPES II [8], where we
showed the first application of NMC controllers on alower
limb exoskeleton. roads, slopes) and walking speed. Also, for people with
some remaining walking capabilities, control would ideally
be shared between the person and the exoskeleton. The
reflex-based musculoskeletal model proposed by Geyer and
colleagues [4], [5] has the potential to tackle both problems. In this model, joint torques are generated by local reflex loops
in such a way that natural gait emerges. The local reflex
loops react to changes in leg motion, for example to devi-
ations due to uneven terrain, or when the person voluntarily
initiates movement by using their remaining motor function. Previously we have incorporated these models as controllers
(called neuromuscular controllers, NMC) for a powered ankle
exoskeleton [6]. In a follow up study, we demonstrated with
this wearable Achilles ankle exoskeleton that incomplete SCI
individuals improved their walking speed and step length
wearing the device over different training sessions, and we
also found an unexpected increased walking speed using no
device after a brief training period with the exoskeleton [7]. NMC controllers for the knee and hips were also rendered
on the haptic robotic gait trainer LOPES II [8], where we
showed the first application of NMC controllers on alower
limb exoskeleton. design (section III), and the low-level and high-level control
(section IV). The device was preliminary evaluated for SCI
individuals with a complete or with an incomplete lesion
(section V). Analogous to those who control prototypes of
airplanes or race cars, we prefer to call our exoskeleton users
SCI test pilots instead of ’individuals’ or ‘patients’ and will
use this term in the remainder of the paper. I. INTRODUCTION van der Kooij is with the Department of Biomechanical Engineering,
Delft University of Technology, 2628 CD Delft, The Netherlands, and also
with the Department of Biomechanical Engineering, University of Twente,
7522 NB Enschede, The Netherlands. H. van der Kooij is with the Department of Biomechanical Engineering,
Delft University of Technology, 2628 CD Delft, The Netherlands, and also
with the Department of Biomechanical Engineering, University of Twente,
7522 NB Enschede, The Netherlands. Traditionally, exoskeletons are controlled using predefined
trajectories, which has resulted in reliable support of people
with a complete lesion during walking over flat ground. However, predefined trajectories are inflexible when it comes
to adapting to varied terrain (e.g., uneven sidewalks, unpaved This article has supplementary downloadable material available at
https://doi.org/10.1109/TNSRE.2021.3049960, provided by the authors. Digital Object Identifier 10.1109/TNSRE.2021.3049960 Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4 s work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativec MEIJNEKE et al.: SYMBITRON EXOSKELETON: DESIGN, CONTROL, AND EVALUATION OF A MODULAR EXOSKELETON 331 TABLE I
PART OF THE USE CASES TABLE FOR THE EXOSKELETON. PRIORITY
IS INDICATED BY M: MANDATORY, D: DESIRED; OR O: OPTIONAL. ACHIEVEMENTS IS INDICATED BY IL: ACHIEVED BY SCI PILOTS
WITH INCOMPLETE LESION, CL: ACHIEVED BY SCI
PILOTS WITH COMPLETE LESION Fig. 1. SCI test pilots using the modular exoskeleton in knee-ankle
configuration (KA) on the left and hip-knee-ankle configuration (HKA) on
the right. TABLE I
PART OF THE USE CASES TABLE FOR THE EXOSKELETON. PRIORITY
IS INDICATED BY M: MANDATORY, D: DESIRED; OR O: OPTIONAL. ACHIEVEMENTS IS INDICATED BY IL: ACHIEVED BY SCI PILOTS
WITH INCOMPLETE LESION, CL: ACHIEVED BY SCI
PILOTS WITH COMPLETE LESION TABLE I Fig. 1. SCI test pilots using the modular exoskeleton in knee-ankle
configuration (KA) on the left and hip-knee-ankle configuration (HKA) on
the right. roads, slopes) and walking speed. Also, for people with
some remaining walking capabilities, control would ideally
be shared between the person and the exoskeleton. The
reflex-based musculoskeletal model proposed by Geyer and
colleagues [4], [5] has the potential to tackle both problems. In this model, joint torques are generated by local reflex loops
in such a way that natural gait emerges. The local reflex
loops react to changes in leg motion, for example to devi-
ations due to uneven terrain, or when the person voluntarily
initiates movement by using their remaining motor function. II. REQUIREMENTS We aimed to develop a personalizable Wearable Exoskeleton
that enables individuals with incomplete or complete SCI to
walk again. The exoskeleton consisted of a set of powered
modules, one for each lower limb joint. Personalization was
accomplished by selecting only those joint modules required
to compensate for the loss of function for that specific per-
sonand tailoring the control and human-machine interface. This paper encloses for the first time the full details about
the mechanical and electrical hardware and on the low level
control of the modular Symbitron exoskeleton, in addition
to CAD drawings of the ankle and knee modules that were
disclosed before [9]. We also show for the first time that
walking speed in incomplete SCI individuals can be increased
with a wearable ankle-knee exoskeleton utilising a biological
inspired neuromuscular control approach. Before starting the design process, the requirements for
the device were specified. First, use cases were specified and
structured
(Table I). Requirements were all rated according
to priority: M Mandatory (essential to function of the device), D Desired (will be aimed for in the design but should not
hinder achieving the mandatory requirements), O Optional (would be nice to have, but no great effort will
be done to achieve it). The table was created from a discussion among clinicians,
researchers, and engineers that were involved in development
of the exoskeleton. The aim of this discussion was to find
a compromise between what is most important for our SCI
test pilots, exceeds or matches the state-of-the-art, and is
technically achievable. In this paper, we describe the use cases and require-
ments for the exoskeleton (section II), the mechatronic IEEE TRANSACTIONS ON NEURAL SYSTEMS AND REHABILITATION ENGINEERING, VOL. 29, 2021 332 Fig. 2. Rendered CAD-model of the Symbitron exoskeleton showing the
joints hip abduction and adduction (HAA), hip internal and external rota-
tion (HIE), hip flexion and extension (HFE), knee flexion and extension
(KFE), ankle dorsiflexion and plantarflexion (ADP), and ankle inversion
and eversion (AIE). The joints with an electricity icon are powered by a
SEA and the joints without are passive. The HIE joint is loaded with an
adjustable spring and can be locked. The AIE joint can be free or locked. This picture shows the hip-knee-ankle (HKA) configuration. The orange
region indicates the right leg Knee-ankle (KA) configuration. The straight
grey arrows show the size adjustments available on the exoskeleton. II. REQUIREMENTS TABLE II
DATA, INCLUDING SPEED IN RPM, TORQUE IN NM, POWER IN W, AND
RANGE OF MOTION (ROM) DEG, FOR VARIOUS USE CASES FROM
TABLE I. BASED ON A MAXIMUM BODY MASS OF OUR SCI PILOTS
OF 85 Kg AND A HEIGHT OF 1.8 m. THE DOTTED VALUES
ARE NOT REPORTED IN THE SOURCE TABLE II DATA, INCLUDING SPEED IN RPM, TORQUE IN NM, POWER IN W, AND
RANGE OF MOTION (ROM) DEG, FOR VARIOUS USE CASES FROM
TABLE I. BASED ON A MAXIMUM BODY MASS OF OUR SCI PILOTS
OF 85 Kg AND A HEIGHT OF 1.8 m. THE DOTTED VALUES
ARE NOT REPORTED IN THE SOURCE From the use cases combined with various data sources,
a table with maximum velocity, torque and power was com-
piled (see Table II). Note that every use case sets additional
design constraints for the system A. Overview Fig. 2. Rendered CAD-model of the Symbitron exoskeleton showing the
joints hip abduction and adduction (HAA), hip internal and external rota-
tion (HIE), hip flexion and extension (HFE), knee flexion and extension
(KFE), ankle dorsiflexion and plantarflexion (ADP), and ankle inversion
and eversion (AIE). The joints with an electricity icon are powered by a
SEA and the joints without are passive. The HIE joint is loaded with an
adjustable spring and can be locked. The AIE joint can be free or locked. This picture shows the hip-knee-ankle (HKA) configuration. The orange
region indicates the right leg Knee-ankle (KA) configuration. The straight
grey arrows show the size adjustments available on the exoskeleton. The exoskeleton has 8 powered joints and 4 passive joints
(Figure 2). Some parts of the exoskeleton can be electrically
and mechanically disconnected from the others and controlled
independently. Due to the position of the attachments to
the user (cuffs, belts, etc.), the following configurations are
possible: • Ankle (A configuration), TABLE III
MEASURED MASS OF THE EXOSKELETON COMPONENTS • Knee-ankle (KA configuration), MEASURED MASS OF THE EXOSKELETON COMPONENTS • Hip-knee-ankle (HKA configuration), • Hip (H configuration). Each configuration can be used for one leg or for both legs. . Each configuration can be used for one leg or for both legs. . The backpack module is always needed because it contains
the control computer, power management, and batteries. When
the hip module is not used, it is worn as a normal backpack
that is not mechanically attached to the knee or ankle modules. Each powered joint contains a custom designed series elastic
actuator (SEA) unit that will be discussed in more detail in
the next section. Table III shows the mass of the exoskeleton
and its sub-assemblies. cylindrical housing with an envelope φ100 × 70 mm. The
main components are: B. The Series Elastic Actuator (SEA) MEIJNEKE et al.: SYMBITRON EXOSKELETON: DESIGN, CONTROL, AND EVALUATION OF A MODULAR EXOSKELETON 333 battery. The power-hungry EtherCAT slaves together with the
internal PC and all other electronics drain the logic battery in
about one hour. In contrast, the motor battery usually lasts for
a multitude of that. • Strain wave gear (Leader drive, LCSG-20). In HKA
configuration, all ratios are chosen to be 100, except
the knee, which is 50. The peak torque and speed are
102 Nm and 3.14 rad/s for gear ratio 100, and 69 Nm
and 6.28 rad/s for ratio 50, respectively. A custom PCB inside the backpack was developed to be
able to switch motor and logic power on or off, and to provide
an extra guard over the battery voltage. This board also
provides: (1) DC power to the PC, EtherCAT splitter , and
IMU interface, and (2) an interface to the emergency stop
button. When the emergency button is pressed, all motor drives
are disabled, and the motor battery is switched off. • Custom design titanium spring. The spring can be loaded
up to 120 Nm with a life of 107 cycles (these specification
were obtained from finite element analysis) and has a
spring constant of 1500 Nm/rad. • Cross-roller bearing (THK, RA 7008). It supports the out-
put rotation including the external and internal moments
in all directions. Computational power is provided by a small PC (Intel
NUC5i5RYH). The communication protocol used in the
exoskeleton is EtherCAT. • Three absolute encoders. These encoders measure the
joint output angle, spring deflection (2 × RLS Aksim
20 bits BiSS), and motor angle (IC-Haus MHM 14 bits
BiSS). Because the purpose of the exoskeleton was enabling
experiments using novel control strategies, it is likely that
interfaces to additional devices are required. Therefore there
is room within the backpack for additional EtherCAT slaves. Several of these were made, and used in different combinations
throughout the experiments: To implement the actuator basis in a joint, the drive elec-
tronics can be oriented and positioned in the most suitable way,
and the housing and output flange are manufactured with the
desired connections to the surrounding parts. The weight of
one actuator including electronics is 1.6 kg. The drive elec-
tronics for each drive consist of a motor drive with EtherCAT
interface (Technosoft, iMOTIONCUBE). A custom EtherCAT
slave was developed to read the joint and spring deflection
encoders, temperature, and an Xsens Inertia Measurement Unit
(IMU). • IMU box. IV. CONTROLLER DESIGN The control PC, situated in the backpack, runs Windows 7. For the EtherCAT master software, TwinCAT 3.1 was chosen
to allow detailed diagnosis of low-level EtherCAT commu-
nication without having to implement additional diagnostics
software. The controllers were developed in Simulink (2015b,
Matlab, Mathworks). The Simulink models were compiled for
real-time usage in TwinCAT. Simulink itself is used as GUI
by running it in ‘external mode’. The model runs at 1000 Hz. The exoskeleton can be tailored to a SCI test pilot by
changing 23 settings (see Figure 2). Most of the components
(such as the bars connecting the knee joint and ankle joint)
are manufactured in multiple sizes and can be interchanged to
change the size of the exoskeleton. This approach was chosen
to reduce weight by omitting telescopic mechanisms and to
create a truly personalized exoskeleton. The control can be separated into two hierarchical lev-
els: low-level and high-level control, which are described
separately below. A. Low-Level Joint Control Low-level control refers to the control of individual joints
to ensure that each joint generates the desired torque on its
output shaft. It includes drive control (turning the drive on
and off), a closed-loop torque controller, software end-stops,
signal logging and safety checks. C. The Structure Apart from the backpack and the actuator electronics,
a small PCB was designed to read the pressure insoles (IEE;
Contern, Luxembourg) and determine heel and toe contact
with the ground. The main goal of the exoskeleton structure is to connect
the actuators to each other and to the human body in such
a way that the actuator’s torque (and external forces) are
conveyed to the wearer in a safe and stable manner. In addition,
the structure facilitates joint-to-joint routing of power and
communication cables. This means that the actuator rotation
center has to coincide with the human joint center as much
as possible. An important constraint for the design of the
structure is to keep the exoskeleton close the body to ensure
that there is minimal interference with the wearer or environ-
ment. As SCI test pilots have different body sizes and shapes,
the exoskeleton structure needs the ability to be resized to each
individual pilot. MEIJNEKE et al.: SYMBITRON EXOSKELETON: DESIGN, CONTROL, AND EVALUATION OF A MODULAR EXOSKELETON This is an interface between the Xbus master
of Xsens (Xsens, Enschede, the Netherlands) and Ether-
CAT. This device makes the quaternion data of 3 IMUs
available on the EtherCAT bus. • Crutch interface. Two buttons were integrated in the
handle of a crutch to initiate steps in experiments with
the HKA configuration. An EtherCAT slave was made to
read those buttons. B. The Series Elastic Actuator (SEA) All actuated joints of the exoskeleton are powered by
a rotary series elastic actuator. The actuator consists of a • Brushless Motor (Tiger-Motor U8 PRO 170Kv). The
maximum continuous output power is 520 W. IEEE TRANSACTIONS ON NEURAL SYSTEMS AND REHABILITATION ENGINEERING, VOL. 29, 2021 334 Fig. 3. Overview of wearable exoskeleton controlled by the NMC. The NMC uses joint kinematics and foot contact measurements to
command joint torques to the device. Stance and swing reflexes are
activated based on contact information.The muscles in the model are
tibialis anterior (TA), soleus (SOL), gastrocnemius (GAS), vasti (VAS),
hamstring (HF), gluteus maximus (GLU), hip flexors (HF), biceps femoris
short head (BFSH), rectus femoris (RF), hip abductors (HAB), and hip
adductors (HAD). joint exerts, τact, is determined by measuring the deflection of
the actuators’ spring. The joint is velocity-limited by a damping controller, which
limits the commanded torque to the motor as τlimited,mot =
min[τdes,mot, −D · (v −vmax)]. Software end stops are
based on the ‘PVA limiter’ as described in [19]. The end
stops were implemented by making the maximum veloc-
ity vmax dependent on the joint position ϕ as vmax(ϕ) =
2 amax(ϕ −ϕendstop). This ensures constant deceleration
(with a = −amax) behavior close to the end stops. Beyond
the end stop (ϕ > ϕendstop), vmax becomes negative to escape
from the region if it was accidentally entered. The main
advantage of this software end stop algorithm over the conven-
tional spring-damper implementation is that the commanded
torque to the motor stays within reasonable values, even when
approaching the end stop with high velocity. Still, the end
stop feels very stiff for low velocities. Also, the penetration
depth beyond the end stop does not depend on the approaching
velocity (and is, theoretically, zero), and it does not suffer from
the ‘sticky effect’. Fig. 3. Overview of wearable exoskeleton controlled by the NMC. The NMC uses joint kinematics and foot contact measurements to
command joint torques to the device. Stance and swing reflexes are
activated based on contact information.The muscles in the model are
tibialis anterior (TA), soleus (SOL), gastrocnemius (GAS), vasti (VAS),
hamstring (HF), gluteus maximus (GLU), hip flexors (HF), biceps femoris
short head (BFSH), rectus femoris (RF), hip abductors (HAB), and hip
adductors (HAD). The end stop behavior takes precedence over the torque
control. Therefore, close to the end stop (and also close to the
maximum allowed velocity), the torque tracking of the joint
may seem poor, but this is because it is more important to
adhere to the end stops (or maximum velocity) to guarantee
safety and to prevent damage to the device than to track the
desired torque well. IEEE TRANSACTIONS ON NEURAL SYSTEMS AND REHABILITATION ENGINEERING, VOL. 29, 2021 versions of the model have previously been implemented and
evaluated on lower limb prostheses [20] and on assistive
devices [6], [8], [21] with promising results for restoring
walking function for impaired individuals. Internally, the NMC consists of multiple muscle reflex
control modules; each of which acts on one or more joints [6]. For the modular exoskeleton, this is an advantage because
it can easily be tailored for the different configurations by
activating only those modules for the joints used. For instance,
for incomplete SCI test pilots
having sufficient hip con-
trol, only the sagittal ankle and knee reflex modules were
activated — modules governing hip movement were excluded. The NMC reacts to the movements of the thighs, resulting
in a synchronized and natural gait. The NMC is nominally
set to provide joint torques for a human simulation model
to walk at 1.3 m/s but can be modified to address specific
SCI test pilot needs. Controller gains, given as a percentage
from zero to hundred percent, modify these nominal torques. Zero gain reduces to minimal impedance mode (a condition
in which the robot seconds user’s intention without delivering
any assistive torques). At the broadest level, controller gains
can be applied symmetrically (to both left and right legs)
or asymmetrically. Joint level gains can also be modified to
augment or reduce the level of assistance at each joint (e.g. the
soleus and tibialis anterior for the ankle). Additionally, each
muscle may be modified individually. For example, the soleus
can be modified separately to yield the level of plantarflexion
needed. A graphic user interface was set up to provide easy
control and modification of controller settings. B. High-Level Control D. The Backpack The electrical components that are not needed on the leg are
placed in the backpack. The description of its contents and the
design choices made are described below. The goal of the torque controller is to command the actuator
to exert a desired torque τdes on its output shaft. The motor
torque required for that, τdes,mot, is determined by a Distur-
bance Observer (DOB) [16], which is an improved version of
the work described in [17] and [18]. The actual torque that the Two Li-ion batteries (Energus; Vilnius, Lithuania) with a
total mass of 2.5 kg are situated in the backpack: one 7S2P
for the logic power
and one 13S3P for the motor power. Interestingly, operation time is entirely limited by the logic B. High-Level Control On top of the low-level controller, a high-level controller
needs to be implemented, which determines how much torque
should be exerted on each of the joints. In order to facilitate
different experiments with the exoskeleton, the high-level con-
troller was implemented as a separate Simulink model which,
after compilation, runs in TwinCAT next to the low-level
controller. Communication between the two was handled by
the TwinCAT ADS server. For each experiment, a separate
Simulink model was developed and loaded into TwinCAT. 1) The Neuromuscular Controller for Incomplete SCI Test
Pilots: The neuromuscular controller (NMC) was based on a
sagittal-plane (2D), reflex-based simulation model developed
by Geyer and Herr [4] and its 3D extension [5] (Figure 3). The
sagittal model is actuated by seven Hill-type muscles per leg to
control the ankle, knee, and hip, including the tibialis anterior,
soleus, gastrocnemius, vasti, hamstring, gluteus maximus, and
hip flexors. The 3D model includes four additional muscles —
biceps femoris short head, rectus femoris, hip abductors, and
hip adductors. Locomotion is produced by simple reflex rules,
which engage depending on whether the leg is in stance or
swing. During stance, the reflexes serve to provide weight sup-
port and to build positive force feedback to prepare for push-
off. During swing, the reflexes utilize flexor muscles to allow
the leg to swing forward and clear the ground. The neuro-
muscular model achieves reasonable predictions of human gait
characteristics such as joint kinematics, kinetic measures, and
muscle activations [4]. It is also robust against perturbations
and environmental disturbances in simulation [5]. Controller 2) Combined Trajectory Controller and NMC for Complete
SCI Test Pilots: Whereas NMC might be very well suited
for individuals with incomplete lesions, the implementation
for individuals with complete lesions faces some challenges. First, initiating gait from stand still cannot yet be appropriately
modelled with NMC. Second, NMC does not directly control
joint trajectories, which could result in potential unsafe tra-
jectories. In order to overcome these challenges for complete MEIJNEKE et al.: SYMBITRON EXOSKELETON: DESIGN, CONTROL, AND EVALUATION OF A MODULAR EXOSKELETON 335 Fig. 4. VI. RESULTS This section shows the results of the experiments described
in the previous section. We will show improvements in walk-
ing speed of the SCI test pilots and the joint kinematics
and kinetics during experiments. But first we will show the
accuracy of the low-level torque tracking since this is a
prerequisite for impedance and NMC controllers. For the combined NMC-trajectory controller, the stiffness
and damping of the trajectory controller and its tunnel width
can be adjusted with the GUI (default: 750 Nm/rad; slightly
overdamped). B. High-Level Control The top panel visualises the concept of the combined NMC and
trajectory controller in which the amount of deviation of the actual joint
angle (red solid line) from the reference joint angle (dashed black line)
determines whether the NMC controller (green region), the trajectory
controller (blue region), or a weighted combination of both (grey area)
is active. In the bottom panel, the relative weighs of the NMC controller
(dashed green line) and trajectory controller (blue solid line) are shown
for this example. TABLE IV
SCI TEST PILOT EPIDEMIOLOGICAL DATA (BOTH HKA
AND KA CONFIGURATION) TABLE IV
SCI TEST PILOT EPIDEMIOLOGICAL DATA (BOTH HKA
AND KA CONFIGURATION) (Table IV; Figure 1 left). The incomplete test pilots (S2 and
S3) used the knee-ankle (KA) configuration of the exoskeleton,
controlled by the NMC. The NMC gains were pre-defined;
the tuning was based on pilot experiments to simultaneously
assess performance and perceived usability. We compared
NMC gait to their shod gait (free walking without wearing the
device). We evaluated the biomechanical gait changes, such as
walking speed, joint kinematics, and joint torques from walk-
ing with the NMC-controlled exoskeleton. For gait analysis for
shod conditions, joint angles and torques were retrieved based
on motion capture data (OptiTrack, NaturalPoint, Corvallis,
OR, recorded at 120 Hz) and ground reaction force data (BTS
Bioengineering, Milan, Italy, recorded at 500 Hz) through
inverse kinematics and dynamics (Opensim [22]). We evalu-
ated the exoskeleton-assisted and shod joint angles and torques
through qualitative comparisons with healthy gait patterns at a
slow speed of 0.6 m/s (Fig. 7). The healthy data was derived
from the average across eight healthy individuals walking on
a treadmill [23]. Fig. 4. The top panel visualises the concept of the combined NMC and
trajectory controller in which the amount of deviation of the actual joint
angle (red solid line) from the reference joint angle (dashed black line)
determines whether the NMC controller (green region), the trajectory
controller (blue region), or a weighted combination of both (grey area)
is active. In the bottom panel, the relative weighs of the NMC controller
(dashed green line) and trajectory controller (blue solid line) are shown
for this example. SCI test pilots, the NMC was combined with an impedance
based trajectory controller. The hip and knee joints were
fully trajectory controlled with a speed-dependent reference
joint trajectory generation algorithm based on the work by
Koopman et al. [13]. B. High-Level Control The algorithm by Koopman generates
reference joint trajectories for continuous walking. For usage
in the exoskeleton, it was adapted to pause after each step so
that the user can shift their weight and adapt their posture to
be ready for the next step. By pressing a button integrated in
the crutch handle, the user could initiate a step that moves one
foot for the other to step or keep on walking, or a step that
put one foot next to the other to end in standing posture. The complete SCI test pilot (S1) used the hip-knee-ankle
(HKA) configuration. Because the complete SCI test pilot was
not able to walk without the exoskeleton, we only show results
of the NMC combined with the trajectory controller. S1 was
also supported by the FLOAT (Lutz Medical Engineering,
Rüdlingen, Switzerland), a body weight support system that
allows overground walking in a rectangular workspace and
safe intervention in case of falls [24]. The ankle joints were controlled by a combination of NMC
and trajectory control (Figure 4): when the joint angle is
close to the reference joint angle as dictated by the (above
mentioned) reference joint trajectory (i.e., within a ‘tunnel’
around the reference trajectory), the NMC is in full control
and the trajectory controller is switched off. When the joint
angle deviates too much from the reference joint angle, the tra-
jectory controller (a proportional-derivative or PD controller to
reduce the error between actual joint angle and reference joint
trajectory) gradually takes over to make sure that the devi-
ation can be limited. This results in natural, NMC-governed
behavior and trajectory-governed behavior if the ankle makes
unexpected movements. Note that the software also allows this
combination of NMC and trajectory control for the knee and
hip joint, but this option was not tested on the SCI test pilots. All SCI test pilots used crutches during the experiments
and gave their informed consent for participation in the
experiments and publication of photos with their image. The
experiments were performed with approval of local ethics
committee, the Comitato Etico Indipendente of Fondazione
Santa Lucia, under approval reference CE/GROG.509. V. PRELIMINARY EVALUATION WITH SCI TEST PILOTS V. PRELIMINARY EVALUATION WITH SCI TEST PILOTS The high-level controller commands desired torques to the
low-level controller, which should then track these torques as
closely as possible. We define torque tracking error as the Walking tests with the exoskeleton were performed by SCI
test pilots with incomplete and complete spinal cord injury IEEE TRANSACTIONS ON NEURAL SYSTEMS AND REHABILITATION ENGINEERING, VOL. 29, 2021 336 Fig. 5. Times series for the joint angle, the actual and desired torques and the torque error of the right leg for two typical strides of complete SCI
test pilot S1. The grey areas indicate the time period in which a joint limiter was active. Fig. 5. Times series for the joint angle, the actual and desired torques and the torque error of the right leg for two typical strides of complete SCI
test pilot S1. The grey areas indicate the time period in which a joint limiter was active. measured actual torque τact minus the commanded torque τdes. Figure 5 shows the torque tracking of two typical strides of
complete SCI test pilot S1. For this test pilot the root mean
square tracking error (RMSE) when the software end-stop was
not active was 0.6, 0.7, 1.4, and 1.8 Nm for the hip abduc-
tion/adduction, hip flexion/extension, knee flexion/extension,
and ankle plantar/dorsal flexion, respectively. well when the software end-stop were not active. Ankle joint
angles were not very well tracked since the NMC controller
was relatively more active than the trajectory controller. We
also collected data after the Symbitron project ended from a
complete SCI individual completing all tasks of the Cybathlon
competition, mostly without body weight support. In the
Supplementary materials, videos and joint angles and torques
from the exoskeleton can be found for standing up and down
and for walking while crossing a tilted path, up and down stairs
and ramps, opening and closing a door, slaloming through
poles, and stepping over stones. By design, sometimes the torque tracking error could be
large. This occurs when the joint nears the software end stop. Then the end stop controller, which minimizes the penetration
beyond end stop, takes precedence over the torque tracking
goal (which minimizes torque error). In Figure 5 the hip
abduction angle approaches its maximal value and so is limited
in two epochs, which is clearly reflected in the drastic increase
of the torque error. C. Incomplete SCI Test Pilots C. Incomplete SCI Test Pilots When used in KA configuration (incomplete SCI pilots
S2 and S3), the NMC-controlled wearable exoskeleton could
support a wide range of walking speeds for each SCI test pilot
(Table V). The pilots were able to dictate the walking speed
by varying their (still intact) thigh behavior; the NMC auto-
matically adjusted the knee and ankle behavior accordingly. B. Complete SCI Test Pilot MEIJNEKE et al.: SYMBITRON EXOSKELETON: DESIGN, CONTROL, AND EVALUATION OF A MODULAR EXOSKELETON Data from healthy persons (dotted)
are included for comparison. Heel-strike at 0%; vertical lines indicate
toe-off. Legend indicates the number of strides over which the mean was
taken. Fig. 6. Speed and stride length for S2 and S3 for the shod (triangle) and
NMC (plus) conditions. Inset: boxplot of walking speeds for S2 and S3. The middle line in the box is the median speed while the bottom and top
edges represent the 25th and 75th percentile, respectively. The whiskers
extend to the minimum and maximum speeds. Strides with stride times
t < 0.8 s and t > 3 s and speeds < 0.2 m/s were discarded to avoid any
turning or very small steps at the beginning or end of trials. profiles with and without robotic assistance were different,
but the NMC-provided torques were closer to natural healthy
torque patterns with extension during the early part of stance
and flexion in late stance. S3’s own knee torques during stance
could arise from the lack of knee flexion during stance. stride length increased with speed (Figure 6). Incomplete SCI
test pilot-specific NMC parameters were kept constant during
all trials, and the NMC successfully supported variations in
walking speed within and among each test pilot without any
pre-defined trajectories. Differing methods for recording kinematics and determining
joint torques could account for some of the disparities between
shod and NMC conditions. For the NMC conditions, exoskele-
ton ankle and knee angles and torques were measured from
the wearable exoskeleton encoders and torque sensors. For the
shod conditions, human joint angles and torques were retrieved
based on motion capture data through inverse kinematics and
dynamics. In particular, the NMC torque is the external torque
exerted by the exoskeleton while the shod torque is the net
joint torque based on a rigid body model through standard
inverse dynamics calculations. NMC provided incomplete SCI test pilots with supporting
torques that resulted in joint kinematics and kinetics that were
similar to healthy gait (Figure 7). Healthy data at a walking
speed of 0.6 m/s was used for qualitative comparisons [23]. Similar to healthy profiles, the NMC provided both complete
SCI test pilots with a peak ankle plantarflexion torque to
assist in ankle push-off and knee extension and flexion torques
during stance. Without the exoskeleton, both S2 and S3 walked
with a more dorsiflexed ankle angle. MEIJNEKE et al.: SYMBITRON EXOSKELETON: DESIGN, CONTROL, AND EVALUATION OF A MODULAR EXOSKELETON 337 TABLE V
RANGE OF WALKING SPEEDS
Fig. 6. Speed and stride length for S2 and S3 for the shod (triangle) and
NMC (plus) conditions. Inset: boxplot of walking speeds for S2 and S3. The middle line in the box is the median speed while the bottom and top
edges represent the 25th and 75th percentile, respectively. The whiskers
extend to the minimum and maximum speeds. Strides with stride times
t < 0.8 s and t > 3 s and speeds < 0.2 m/s were discarded to avoid any
turning or very small steps at the beginning or end of trials. stride length increased with speed (Figure 6). Incomplete SCI
test pilot-specific NMC parameters were kept constant during
all trials and the NMC successfully supported variations in
Fig. 7. Mean joint angles and torques for the right ankle and right knee
for S2 and S3 as a function of stride. Walking conditions include shod
(dashed or double-dashed) and NMC (solid or solid-dotted) with two dif-
ferent speeds (fast: dark, slow: light). Data from healthy persons (dotted)
are included for comparison. Heel-strike at 0%; vertical lines indicate
toe-off. Legend indicates the number of strides over which the mean was
taken. profiles with and without robotic assistance were different,
but the NMC-provided torques were closer to natural healthy
torque patterns with extension during the early part of stance
and flexion in late stance. S3’s own knee torques during stance
could arise from the lack of knee flexion during stance. TABLE V
RANGE OF WALKING SPEEDS TABLE V
RANGE OF WALKING SPEEDS Fig. 6. Speed and stride length for S2 and S3 for the shod (triangle) and
NMC (plus) conditions. Inset: boxplot of walking speeds for S2 and S3. The middle line in the box is the median speed while the bottom and top
edges represent the 25th and 75th percentile, respectively. The whiskers
extend to the minimum and maximum speeds. Strides with stride times
t < 0.8 s and t > 3 s and speeds < 0.2 m/s were discarded to avoid any
turning or very small steps at the beginning or end of trials. Fig. 7. Mean joint angles and torques for the right ankle and right knee
for S2 and S3 as a function of stride. Walking conditions include shod
(dashed or double-dashed) and NMC (solid or solid-dotted) with two dif-
ferent speeds (fast: dark, slow: light). MEIJNEKE et al.: SYMBITRON EXOSKELETON: DESIGN, CONTROL, AND EVALUATION OF A MODULAR EXOSKELETON With the NMC, the ankle
angles of both test pilots were less dorsiflexed and closer to
normative ankle patterns. For S2, knee angles with NMC were
similar in profile with shod condition and healthy data but
with less knee flexion during swing. For S3, the knee torque VII. DISCUSSION B. Complete SCI Test Pilot For S1, the complete SCI test pilot was able to walk
with the combined NMC and trajectory controller with an
average speed of 0.1 m/s (Table V.) Torques exerted by the
exoskeleton are shown in Figure 5. The joint angles of the
hip and knee tracked the reference joint angles reasonably Both test pilots walked faster with the aid of the robot
than without it. The NMC allowed the SCI test pilots to
change their walking speed and stride length. The individual’s MEIJNEKE et al.: SYMBITRON EXOSKELETON: DESIGN, CONTROL, AND EVALUATION OF A MODULAR EXOSKELETON IEEE TRANSACTIONS ON NEURAL SYSTEMS AND REHABILITATION ENGINEERING, VOL. 29, 2021 IEEE TRANSACTIONS ON NEURAL SYSTEMS AND REHABILITATION ENGINEERING, VOL. 29, 2021 338 TABLE VI
COMPARISON OF MAIN CHARACTERISTICS FOR
VARIOUS EXOSKELETONS∗ TABLE VI
COMPARISON OF MAIN CHARACTERISTICS FOR
VARIOUS EXOSKELETONS∗ lesion levels and gradations. We have developed a modular
system that can be customized for each person by selecting the
relevant joint modules and by adjusting the 23 length settings
appropriately. Experiments were done with different configurations: HKA
(Hip/Knee/Ankle) and KA (Knee/Ankle) on test pilots with
different impairment levels. For people who still had remaining
walking ability , we found an increased walking speed when
wearing the exoskeleton. The NMC controller enabled the
exoskeleton to automatically adapt to changes in walking
velocity. The emerging gait and torques were mostly similar
to those found in healthy people. People who fully lost their
walking ability could also walk again when wearing the
exoskeleton. advantage of using SEA is to have much better force control
capacity, which is essential for implementing bio-inspired
controllers and for partial support of gait and balance. Most
lower-leg exoskeletons for SCI do not actuate the ankle, while
we do. An ankle-only exoskeleton evaluated in individuals
with SCI showed improved push off kinematics and a small
reduction of activity in muscles involved in push off [27]. We recently found similar advantages of such a device [7]. IHMC’s Mina v2 exoskeleton [28] also includes actuation of
the ankle. In their report about IHMC’s participation in the
Cybathlon 2016, the authors state that the addition of the
powered ankle to Mina v2 indeed propelled the patient forward
during walking and made walking more stable. Also, in the
Atalante exoskeleton, active control of the ankle is necessary
to maintain balance. So, inclusion of ankle actuation holds
promise to improve walking efficiency in individuals with SCI
and is essential to include for supporting balance. We conclude from these preliminary results that the NMC
can support gait for a large range of speeds and is capable
of generating healthy-like gait for SCI test pilots with various
levels of mobility during overground walking. In particular,
for S2 and S3, NMC ankle and knee torque profiles were
similar to healthy ones. While there were some differences
in joint angles, the NMC produced more normative ankle
angles than the SCI test pilots could achieve without the aid
of the exoskeleton. B. Future Work Future development for the exoskeleton will be aimed at
reducing weight, increasing robustness, and increasing main-
tainability and further developing high level control software. Weight reduction was a major goal in design of the actu-
ators. The structure, however, was harder to optimize due
to the many available size-adjustments of the exoskeleton. Additionally, its modularity needs to be reconsidered because
it increases the number of interfaces even further. Future
development will be aimed at finding fast and low-cost ways
to produce a one-to-one structure for each SCI test pilot to
minimize mass and volume. Also the weight of the backpack
will be reduced significantly. Large sources of failure in the exoskeleton were the cables
and connectors. Again, because of the number of available size
adjustments, the cables were running external to the exoskele-
ton structure, creating vulnerabilities. Here, modularity also
creates additional complexity by demanding an additional con-
nection and requiring variable cable length. More effort needs
to be made in the integration of the cables and connectors
in the structure. Finally, design for maintenance was greatly
underestimated in the project. In future designs, electronics
should be accessible and easily replaced. IEEE TRANSACTIONS ON NEURAL SYSTEMS AND REHABILITATION ENGINEERING, VOL. 29, 2021 Using only a fixed set of controller gains
tailored to each user’s needs, the diversity of gaits achieved
demonstrates the versatility and capability of a bio-inspired
controller. The modularity of this controller is also particularly
suitable for use in a modular exoskeleton, such as the one
detailed in this paper. From the results we presented and
data in the supplementary materials, it is evident that for the
incomplete SCI test pilots all requirements were met, while
for complete SCI test pilots all mandatory requirements were
achieved (see Table I). In addition to the experiments presented in this paper,
we also used the exoskeleton to support standing balance
in our SCI test pilots [25]. Using the exoskeleton in the
ankle-knee configuration, we tested how various low-level con-
trollers (PD control on center of mass and a momentum-based
controller [26]) performed in assisting individuals with incom-
plete SCI with maintaining their balance while standing. The
centre of mass was estimated from the joint angles of the
exoskeleton and IMUs attached to the chest and right thigh. Results were promising since all SCI test pilots improved
their balance recovery after being perturbed. One of the
SCI test pilots could only stand stably with help of the
exoskeleton [25]. VII. DISCUSSION Our aim was to develop an exoskeleton for individuals
with a SCI that can be personalized to a broader range of ACKNOWLEDGMENT The authors would like to specially thank Wouter Gregoor,
Menno Lageweg (TUDelft), and Quint Meinders (UTwente)
for helping with design and realization of the exoskeleton and
the excellent support during the experiments. [16] W. F. Rampeltshammer, A. Q. L. Keemink, and H. van der Kooij,
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A novel bioinspired framework using motor primitives for locomotion
assistance through a wearable cooperative exoskeleton,” IEEE Robot. Autom. Mag., vol. 23, no. 1, pp. 83–95, Mar. 2016. [5] S. Song and H. ACKNOWLEDGMENT Geyer, “A neural circuitry that emphasizes spinal feed-
back generates diverse behaviours of human locomotion,” J. Physiol.,
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and A. J. Ijspeert, “Mechanics of very slow human walking,” Sci. Rep.,
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gait training,” in Proc. IEEE 13th Int. Conf. Rehabil. Robot. (ICORR),
Jun. 2013, Art. no. 6650512. [7] F. Tamburella et al., “Neuromuscular controller embedded in a powered
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perspective of incomplete spinal cord injured subjects,” IEEE Trans. Neural Syst. Rehabil. Eng., vol. 28, no. 5, pp. 1157–1167, May 2020. [25] A. Emmens et al., “Improving the standing balance of paraplegics
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humanoid robot: Structure and properties,” in Proc. IEEE/RSJ Int. Conf. Intell. Robots Syst., Sep. 2008, pp. 653–659. [9] C. Meijneke, S. Wang, V. Sluiter, and H. van der Kooij, “Introducing
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peak kinematic and kinetic parameters from gait speed,” Gait Posture,
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dependent reference joint trajectory generation for robotic gait support,”
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vol. 39, no. 9, pp. 1621–1628, Jan. 2006. [15] S. Nadeau, B. J. McFadyen, and F. Malouin, “Frontal and sagittal
plane analyses of the stair climbing task in healthy adults aged over
40 years: What are the challenges compared to level walking?” Clin. Biomechanics, vol. 18, no. 10, pp. 950–959, Dec. 2003. ACKNOWLEDGMENT Lankhorst, “Biomechanics and muscular activity during sit-to-stand
transfer,” Clin. Biomechanics, vol. 9, no. 4, pp. 235–244, Jul. 1994. p
[28] R. Griffin et al., “Stepping forward with exoskeletons: Team IHMC? s
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vol. 24, no. 4, pp. 66–74, Dec. 2017.
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A Functional Analysis of the Finnish 2012 Presidential Elections
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1. Introduction Presidential elections are one of the most anticipated and high-profile events of a democratic society: Every few years,
the people gather together and express their opinion on who should be the leader of their nation for the next several
years. Presidential candidates do their best to distinguish themselves from one another and to present themselves in a
positive light, and the people will try to make an informed decision on who, from their personal perspective, is the best
candidate for the job. In making this important decision they are influenced by different forms of campaign messages
that aim not only to provide information, but also to influence the voters’ final decision. Perhaps the most important of
these message forms is presidential debates. Televised debates are extremely important (Benoit, 2007, 2014a, 2014b) due to their various advantages compared to
other campaign message forms. Compared to, for example, a television spot or an advertisement aired on radio channels,
televised debates give the candidates much more room to present their case and to distinguish themselves from one
another. In debates, candidates are engaged in dialogue, which makes it easier for the voters to make comparisons
between the candidates. Since candidates are usually not allowed to bring any notes to these debates, they offer voters a
chance to see a more spontaneous side of them. Finally, debates usually generate a lot of attention both from media and
from general public, which means increased public discussion that ultimately benefits the voters (Benoit, 2007, 2014a,
2014b). Several studies on the effects of watching televised debates indicate that not only does watching these debates
increase the knowledge of the voters, but also has the capability of affecting their final voting decisions, especially in
cases where they were originally undecided (e.g., Benoit, Hansen, & Verser, 2003; Lemert, 1993; McKinney & Warner,
2013; Pfau, 2002; Schrott, 1990). Therefore, it is clear that televised presidential elections merit scholarly attention. In this research the functional theory of political campaign discourse, developed by Benoit and his colleagues (e.g.,
Benoit, 2007, 2014a, 2014b; Benoit, Blaney, & Pier, 2000), will be applied to analyse the televised presidential debates
of the 2012 presidential elections in Finland. Abstract This study applied the functional theory of political campaign discourse, developed for political campaigns in the United
States to two televised presidential debates in the 2012 presidential elections in Finland. Acclaims were the most
preferred statement by the candidates, with agreements being the least preferred. Policy was discussed more than
character during the debates. General goals and ideals were used more frequently to acclaim than to attack. Results are
generally consistent with the results of previous studies of presidential elections in the US and other countries. However,
differences did emerge: the classical functional categories were supplemented by a new category, the role of the
moderator as an attacker in the debate is emphasized, the significance of the diminishing role of the Finnish Presidency
is of significance, and the fact that one of the two candidates was the first openly homosexual presidential candidate
likely influenced the debates and the election. Keywords: functional theory, political communication, presidential debates, chi-square This is an electronic reprint of the original article.
This reprint may differ from the original in pagination and typographic detail.
Author(s):
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A Functional Analysis of the Finnish 2012 Presidential Elections
Paatelainen, Laura; Croucher, Stephen; Benoit, Bill
Paatelainen, L., Croucher, S., & Benoit, B. (2016). A Functional Analysis of the Finnish
2012 Presidential Elections. Studies in Media and Communication, 4(2), 70-80.
https://doi.org/10.11114/smc.v4i2.1826
2016 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s):
Title:
Year:
Version:
Please cite the original version:
A Functional Analysis of the Finnish 2012 Presidential Elections
Paatelainen, Laura; Croucher, Stephen; Benoit, Bill
Paatelainen, L., Croucher, S., & Benoit, B. (2016). A Functional Analysis of the Finnish
2012 Presidential Elections. Studies in Media and Communication, 4(2), 70-80. https://doi.org/10.11114/smc.v4i2.1826
2016 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s):
Title:
Year:
Version:
Please cite the original version:
A Functional Analysis of the Finnish 2012 Presidential Elections
Paatelainen, Laura; Croucher, Stephen; Benoit, Bill
Paatelainen, L., Croucher, S., & Benoit, B. (2016). A Functional Analysis of the Finnish
2012 Presidential Elections. Studies in Media and Communication, 4(2), 70-80. https://doi.org/10.11114/smc.v4i2.1826
2016 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Please cite the original version:
Paatelainen, L., Croucher, S., & Benoit, B. (2016). A Functional Analysis of the Finnish
2012 Presidential Elections. Studies in Media and Communication, 4(2), 70-80. https://doi.org/10.11114/smc.v4i2.1826 All material supplied via JYX is protected by copyright and other intellectual property rights, and
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offered, whether for sale or otherwise to anyone who is not an authorised user. Studies in Media and Communication
Vol. 4, No. 2; December 2016
ISSN 2325-8071 E-ISSN 2325-808X
Published by Redfame Publishing
URL: http://smc.redfame.com Studies in Media and Communication
Vol. 4, No. 2; December 2016
ISSN 2325-8071 E-ISSN 2325-808X
Published by Redfame Publishing
URL: http://smc.redfame.com Received: August 22, 2016 Accepted: September 7, 2016 Online Published: September 21, 2016
doi:10.11114/smc.v4i2.1826 URL: http://dx.doi.org/10.11114/smc.v4i2.1826 Received: August 22, 2016 Accepted: September 7, 2016 Online Published: September 21, 2016
doi:10.11114/smc.v4i2.1826 URL: http://dx.doi.org/10.11114/smc.v4i2.1826 Received: August 22, 2016 Accepted: September 7, 2016 Online Published: September 2
doi:10.11114/smc.v4i2.1826 URL: http://dx.doi.org/10.11114/smc.v4i2.1826 A Functional Analysis of the Finnish 2012 Presidential Elections Laura Paatelainen1, Stephen Croucher2, Bill Benoit3
1Doctoral Student in Speech Communication at the University of Tampere, Finland
2Professor of Intercultural Communication at the University of Jyväskylä, Finland
3Professor of Communication at Ohio University, USA
Correspondence: Stephen Croucher, Professor of Intercultural Communication at the Univer 1. Introduction Functional Theory makes a series of assumptions and predictions on the
utterances performed by candidates in their campaign discourse and has been used to analyse presidential debates in the
United States (Benoit, 2014b), among other forms of campaign messages, which include television spots, direct mail
advertising and talk show appearances. The theory has also been applied to presidential elections in other countries with
mixed results: most studies seem to indicate presidential campaign discourse is the same across borders and cultures, 70 Studies in Media and Communication Vol. 4, No. 2; 2016 yet a few studies (e.g., Cmeciu & Patrut, 2010; Hrbková & Zagrapan, 2014; Isotalus, 2011) have offered criticism of the
theory, claiming it to be too culturally limited to be useful in cultures different from the United States. Isotalus (2011)
applied the functional theory to Finnish elections, analysing the 2006 presidential elections in Finland. He criticized the
theory for not being applicable in multi-party systems and for not being suitable for analysing political campaign
discourse in Finland, as the Finnish speech culture differs greatly from the American speech culture. It is possible that
cultural differences influence the content of political leaders’ debates. However, Functional Theory has been
successfully applied to debates in multi-party systems: Australia, Canada, Northern Ireland, Scotland, the United
Kingdom, and Wales (see Benoit, 2014b). Even though the functional theory of political campaign discourse has been applied to Finnish presidential debates,
there are strong reasons for conducting another study. First, applying the theory to another set of presidential debates in
the same country helps to determine whether the results of the first study are really caused by cultural differences or
whether they were only applicable to one set of debates. Second, the Finnish political system offers an interesting
opportunity to study political leaders’ debates. In the United States the president is clearly in charge of running the
government; however, in Finland the president has in recent years been stripped from a large amount of political power. Instead of running the government, the president is seen as a “symbol of the nation”, somebody who represents the core
values of the country and is in charge of international relations of the country (Halonen, 2002) – again, symbolically, as
for example the matters concerning the European Union are mainly handled by the Prime Minister and the government. 1. Introduction Third, the 2012 presidential elections in Finland were particularly interesting and deserve a closer analysis, which has
not been provided so far. In the 2012 elections there was no incumbent candidate, as President Tarja Halonen was
leaving office. Out of the eight candidates, Mr. Sauli Niinistö was predicted by many as the clear winner (Kinnunen,
2011). Nevertheless, the elections preceded to a second round, where Mr. Niinistö was challenged by Mr. Pekka
Haavisto – the first openly homosexual candidate in the history of Finland. In the end, the 2012 presidential elections
was an election of values and ideals. Many voters, especially younger ones, voted for Mr. Haavisto simply for the
reason of wanting to support gay rights in Finland. Some people also voted Mr. Niinistö for the same reason: They did
not want to have a homosexual president in Finland (Blencowe, 2012). Therefore, although one of the assumptions
made by the functional theory of political campaign discourse is that in presidential elections, policy issues matters
more than the character of the candidates; in this particular set of elections it is clear that character and personal
attributes played a decisive role. The research reported here will serve as an analysis of the political campaign discourse in the context of Finnish
presidential elections. In addition to that, it will also serve as a cultural comparison, where the results of this Finnish
analysis will be compared to those of studies conducted in the United States and other countries. 2. Literature Review A different result was reached in the Ukraine (Benoit & Klyukovski, 2006) where attacks were more
common than acclaims; however, this unusual result is at least partly due to the exceptional nature of this Ukrainian
campaign (the campaign included voter fraud as well as accusations of one candidate poisoning the other). applied to several elections in various countries outside of the US. So far, functional theory has been used to analyse
political campaign discourse in Slovakia (Hrbková & Zagrapan, 2014), the United Kingdom (Benoit & Benoit-Bryan,
2013), France (Choi & Benoit, 2013), Spain (Herrero & Benoit, 2009), Israel (Benoit & Sheafer, 2006), Taiwan (Benoit,
Wen, & Yu, 2007), Romania (Cmeciu & Patrut, 2010), Ukraine (Benoit & Klyukovski, 2006), Germany (Benoit &
Hemmer, 2007), Korea (Lee & Benoit, 2004, 2005), Finland (Isotalus 2010, 2011), Canada (Benoit, 2011; Benoit &
Henson, 2007), and Australia (Benoit & Henson, 2007). Applying functional theory to research the political campaign
discourse in countries other than the US has raised the question of whether the theory is too culturally limited to be
useful in political systems different from that of the US. Critique towards the functional theory has been brought
forward by Isotalus (2010, 2011) who claims, first, the theory was developed to be used in a two-party system and is
therefore difficult to apply to a multi-party system, second, the functional theory only works in elections where the
character of the candidate is important (Isotalus & Aarnio, 2005 in Isotalus, 2011) and third, some forms of political
discourse are culturally bound and therefore the division to attacks, acclaims and defences is not flexible enough to
analyse political campaign debates in all cultures. Cmeciu and Patrut (2010) agreed with this critique, arguing that
political campaign discourse is not consistent across borders and cultures; indeed, their study of the 2009 Romanian
presidential debates revealed the debates were not focused on acclaims and policy, as argued by the functional theory,
but instead on attacks and defences. Also Hrbková and Zagrapan’s (2014) research of the 2012 election debates in
Slovakia reached similar conclusions, arguing the categories of content analysis should be expanded as with the current
categories more than 30 percent of the content of the debates would be excluded from the analysis – an argument also
made by Isotalus (2011). 2. Literature Review Nevertheless, from the content analysed, acclaims were still the most common category,
followed by attacks, which means the results reached in Slovakia are at least somewhat similar to those reached in the
United States. A different result was reached in the Ukraine (Benoit & Klyukovski, 2006) where attacks were more
common than acclaims; however, this unusual result is at least partly due to the exceptional nature of this Ukrainian
campaign (the campaign included voter fraud as well as accusations of one candidate poisoning the other). In Finland, televised presidential debates – sometimes called discussions in the media – are still a relatively
understudied phenomenon. Research has been done on argumentation in presidential debates (Kaija & Malinen, 2007),
communication style (Kuivasmäki, 2000; Tiittula, Nuolijärvi & Isotalus, 2007), and constructing the candidate’s
identities (Halonen, 2000). Isotalus (2009, 2011) applied the functional theory of political campaign discourse to the
analysis of Finnish presidential debates in 2006; he stated that although generally the results correspond to the results
found in US, the theory itself is not a suitable tool for analysing Finnish presidential debates, as so many utterances are
left unanalysed. Nevertheless, with so little attention being given to content analysis of Finnish presidential debates,
another look is warranted. 3. Theoretical Underpinning The functional theory of political campaign discourse, developed in the US by Benoit (2007, 2014a, 2014b; Benoit et al.,
1998, 2002, 2003) provides the theoretical foundation for this study. The theory is based on the idea that political
campaign messages are always inherently functional in their very nature, as they are delivered to achieve one purpose:
the winning of elections. This is most likely true in two-party systems, where both candidates have a reasonable chance
of winning the debate; however, as acknowledged by Benoit (2007), it is possible that sometimes in the elections there
are candidates who do not stand a chance of winning the elections and who therefore use the campaign to fulfil some
other purpose, such as laying groundwork for the next elections or furthering the agenda of their own party. This theory
presents five different assumptions or axioms that lay the groundwork for the theory: first, voting is a comparative act,
second, candidates must distinguish themselves from their opponents in a positive light, third, political campaign
messages allow candidates to distinguish themselves, fourth, candidates establish preferability through acclaiming,
attacking and defending, and fifth and finally, campaign discourse occurs on two topics: policy and character. The
underlying idea is that candidates can only seem preferable to other candidates if they seem different – if all the
candidates were indistinguishable, no one would know who to vote for, and the voter turnout would probably hit record
lows. Candidates can make themselves look more preferable either by highlighting their own strengths (by acclaiming
and defending) or pointing out the weaknesses of their opponents (by attacking). This can happen either on the level of
policy (governmental action and problems amenable to such an action) or character (the characteristic or qualities of the
candidates.) Both policy and character are then divided into three further categories for closer analysis: policy can be
discussed either on the level of past deeds, general goals or future plans, and character can focus on personal qualities,
leadership abilities, and ideals. This study tests four hypotheses derived from the functional theory and confirmed
through previous research (e.g., Benoit & Benoit-Bryan, 2013; Benoit, Delbert, Sudbrock, & Vogt, 2010; Benoit et al.,
2011; Benit, Henson, Davis, Glantz, Phillips, & Rill, 2013; Brazeal & Benoit, 2001, 2006). 2. Literature Review This section presents a review of the research on televised political leaders’ debates. A large portion of the research is
focused on analysing presidential elections in the US. This US-focused research includes analysis of the language and
rhetoric of presidential debates (e.g. Cienki, 2004; Halmari, 2008; Peifer & Holbert, 2013; Rhea, 2012), argumentation
strategies (e.g. Hollihan, 2009; Roitman, 2015; Straub, Beller, & Hunt, 2012; Zarefsky, 2008;), issue ownership (e.g. Benoit & Hansen, 2004; Cole & Hawthorne, 2013) and effects of political leaders’ debates on issue knowledge and
voter behaviour (e.g., Benoit & Hansen, 2004; Benoit et al., 2003; Benoit, McKinney & Stephenson, 2006; Pfau, 2002). According to these studies televised debates increase issue knowledge and influence voters’ perception of the candidates
and voter behaviour, especially in situations where voters were undecided before watching the debates. Benoit’s (e.g., 2007, 2014a, 2014b; Benoit, Webber, & Berman, 1998; Benoit, Pier, Brazeal, McHale, Klyokovski, &
Airne, 2002; Benoit et al., 2003) functional theory on political campaign discourse is one of the most used theories in
research of televised political leaders’ debates. The theory is focused on analysing the content of the campaign messages
and classifying that content into attacks, acclaims, and defences – as well as into policy and character utterances
–according to what the candidates said, thus resulting in better understanding of ”tactics” employed by the campaigning
politicians. The functional theory has been applied to many different kinds of campaign messages in the US, including
debates ranging from presidential primary debates, general election debates, vice-presidential debates, senate debates,
gubernatorial debates, and mayoral debates (see Benoit, 2014b). The results of these studies are similar: in presidential
election debates, acclaims are generally used more than attacks, which are used more than defences, and policy is
discussed more than character. The challengers use more attacks than incumbents, who are more prone to using
acclaims (Benoit, 2014b). Also the results of research on other campaign mediums – web pages, radio spots, television
spots, talk show appearances, and convention speeches – seem to follow a similar pattern (Benoit, 2007). The functional theory of political campaign discourse was originally designed to analyse election campaigns in the
United States (Benoit, 2007). In recent years, however, the functional theory of political campaign discourse has been 71 Studies in Media and Communication Vol. 4, No. 2; 2016 applied to several elections in various countries outside of the US. 2. Literature Review So far, functional theory has been used to analyse
political campaign discourse in Slovakia (Hrbková & Zagrapan, 2014), the United Kingdom (Benoit & Benoit-Bryan,
2013), France (Choi & Benoit, 2013), Spain (Herrero & Benoit, 2009), Israel (Benoit & Sheafer, 2006), Taiwan (Benoit,
Wen, & Yu, 2007), Romania (Cmeciu & Patrut, 2010), Ukraine (Benoit & Klyukovski, 2006), Germany (Benoit &
Hemmer, 2007), Korea (Lee & Benoit, 2004, 2005), Finland (Isotalus 2010, 2011), Canada (Benoit, 2011; Benoit &
Henson, 2007), and Australia (Benoit & Henson, 2007). Applying functional theory to research the political campaign
discourse in countries other than the US has raised the question of whether the theory is too culturally limited to be
useful in political systems different from that of the US. Critique towards the functional theory has been brought
forward by Isotalus (2010, 2011) who claims, first, the theory was developed to be used in a two-party system and is
therefore difficult to apply to a multi-party system, second, the functional theory only works in elections where the
character of the candidate is important (Isotalus & Aarnio, 2005 in Isotalus, 2011) and third, some forms of political
discourse are culturally bound and therefore the division to attacks, acclaims and defences is not flexible enough to
analyse political campaign debates in all cultures. Cmeciu and Patrut (2010) agreed with this critique, arguing that
political campaign discourse is not consistent across borders and cultures; indeed, their study of the 2009 Romanian
presidential debates revealed the debates were not focused on acclaims and policy, as argued by the functional theory,
but instead on attacks and defences. Also Hrbková and Zagrapan’s (2014) research of the 2012 election debates in
Slovakia reached similar conclusions, arguing the categories of content analysis should be expanded as with the current
categories more than 30 percent of the content of the debates would be excluded from the analysis – an argument also
made by Isotalus (2011). Nevertheless, from the content analysed, acclaims were still the most common category,
followed by attacks, which means the results reached in Slovakia are at least somewhat similar to those reached in the
United States. 4. Method This study analysed two Finnish presidential debates from 2012. Both of these debates took place in the second round
of the 2011 presidential elections. Participants included the two remaining presidential candidates, Mr. Sauli Niinistö of
the Coalition Party and Mr. Pekka Haavisto of the Green Party, as well as two moderators. The debates took place on
January 26, 2012 and February 2, 2012 and were broadcast by YLE (Finnish public service broadcasting company). Both of the debates lasted an hour. There were also other televised debates arranged by other broadcasting companies;
the debates broadcast by YLE were chosen because of the company’s nature as a public service – and thus, deemed to
be most objective – company. Debates also took place in the first round of the presidential elections with all the initial
eight candidates; however, a decision was made to focus on the second round debates as they, with two remaining
candidates, resembled more closely the format of the American presidential election debates. Due to this resemblance, it
was possible to look past the differences between political systems (two-party vs. multi-party systems) and focus on the
possible cultural differences between Finnish and American debates. The data was analysed applying the same procedures used in previous studies using functional theory (e.g. see Benoit,
2007, 2014a, 2014b) and statistical significance was tested with chi-square test. The texts of these debates were divided
into themes. Themes are complete ideas, arguments, or claims capable of expressing different functions. The length of a
theme can vary from a single phrase to several sentences. Once the themes were identified, they were categorised by
function: acclaims, attacks, defences, and agreements. Next, the themes were classified by topic: policy or character. Finally, policy utterances were divided further into utterances concerning general goals, past deeds, and future plans,
and character utterances were divided personal qualities, leadership abilities, and ideals. The original method was modified slightly to take into account some characteristics of these televised debates. Originally, the functional theory only consisted of three categories: acclaims, attacks, and defences. In this particular
study, a fourth category called agreements was added. Isotalus (2011) claimed agreements are a characteristic typical to
Finnish presidential debates; this category was added to examine this claim further. Second, the original method does
not take into account the utterances made by the moderators as they do not play a meaningful role in the debates. 3. Theoretical Underpinning According to the functional theory, acclaims are the “safest choice” for candidates: they highlight the best qualities of
the candidates without having any visible drawbacks (this does not mean all acclaims are persuasive, just that acclaims 72 Vol. 4, No. 2; 2016 Studies in Media and Communication have fewer drawbacks than the other two functions). Attacks can be useful in highlighting the weaknesses of other
candidates, yet they have their dangers too: studies have shown voters dislike “mud-slinging,” so too many attacks or
badly timed attacks may cause the voters to turn on the candidate making these attacks. Compared to the other two,
defences are said to be the least useful function: Although they can help to reduce damage made by an attack or to
restore the candidate’s damaged image (Benoit, 2007), they also force the candidates to draw more attention to the
attack in the first place, reminding the voters of the attack. They also prevent the candidates from using the time for
other, more beneficial utterances, such as highlighting their strengths by acclaiming. Thus, based on theory and research
about the function of acclaims and defences, the first hypothesis is proposed: have fewer drawbacks than the other two functions). Attacks can be useful in highlighting the weaknesses of other
candidates, yet they have their dangers too: studies have shown voters dislike “mud-slinging,” so too many attacks or
badly timed attacks may cause the voters to turn on the candidate making these attacks. Compared to the other two,
defences are said to be the least useful function: Although they can help to reduce damage made by an attack or to
restore the candidate’s damaged image (Benoit, 2007), they also force the candidates to draw more attention to the
attack in the first place, reminding the voters of the attack. They also prevent the candidates from using the time for
other, more beneficial utterances, such as highlighting their strengths by acclaiming. Thus, based on theory and research
about the function of acclaims and defences, the first hypothesis is proposed: H1: Acclaims will be the most common function and defences will be the least common function in the 2012 Finnish
presidential debates. Previous research shows that in American presidential elections policy is discussed more than character (even 75% to
25%). This result has also been confirmed in other countries (e.g., Choi & Benoit, 2013). 3. Theoretical Underpinning Therefore, based on the
research suggesting differences in the frequency of policy and character discussion the second hypothesis is put forth: H2: Policy will be discussed more than character in the 2012 Finnish presidential debates. The reasoning for both hypotheses 3 and 4 is the same: General goals and ideals are both vague and unspecific in their
very nature, so attacking them is more difficult than attacking for example very specifically laid out future plans. It can
be difficult, and even harmful, to disagree with general goals such as “I want to reduce poverty in this country” or with
ideals such as “I believe in equality”, as the general population sees reducing poverty and equality as positive ideas. For
this reason, both general goals and ideals are considered to be “safe” tools for acclaiming: the likelihood of backlash is
relatively small, since attacking generally accepted ideas would be most likely to hurt the attacking candidate than the
candidate being attacked. These results have been confirmed in previous research (e.g. Benoit 2007, 2011; Benoit &
Benoit-Bryan, 2013). Therefore, based on previous research, the following two hypotheses are presented: H3: General goals will be used more frequently to acclaim than to attack in the 2012 Finnish presidential debates. H3: General goals will be used more frequently to acclaim than to attack in the 2012 Finnish president H4: Ideals will be used more frequently to acclaim than to attack in the 2012 Finnish presidential debat als will be used more frequently to acclaim than to attack in the 2012 Finnish presidential debates. Originally, the functional theory of political campaign discourse also included two other hypotheses. Both of these
hypotheses concern the role of the incumbent candidate in the debates. Since in the 2012 Finnish presidential elections
there was no incumbent candidate, these two hypotheses (hypotheses 3 and 4 in the original theory) were not included
in the analysis. 5. Context of the 2012 Finnish Debates Despite this, in the first round he received less than 50
percent of all votes, which meant the elections proceeded to the second round. In the first round, Mr. Niinistö got 37
percent of the votes, followed by Mr. Pekka Haavisto from the Green Party (18.8 %) and Mr. Paavo Väyrynen from the
Centre Party (17.5%). The other candidates - Mr Timo Soini from True Finns (9.4%), Mr Paavo Lipponen from the
Social Democratic Party of Finland (6.7%) Mr. Paavo Arhinmäki from the Left Alliance (5.5%), Mrs. Eva Biaudet from
Swedish People’s Party of Finland (2.7%) and Mrs. Sari Essayah from the Christian Democrats (2.5%) – were left far
behind (Statistics Finland, 2012). In the second round of the elections, Mr. Niinistö was challenged by Mr. Haavisto, but
still managed to win the elections as expected with a clear result, 62.6 percent of all votes compared to Mr. Haavisto’s
37.4 percent. In the 2012 presidential campaign there were two distinct features. First, Mr. Sauli Niinistö was a clear favourite
throughout the whole campaign – so much so that it most likely affected the overall campaign. It is likely that with such
a clear winner, most of the other candidates were not really campaigning with the goal of winning the election, but had
other aims in mind, such as gathering more support for their party or laying the groundwork for future elections. It has
also been noted that in the first round of the election debates, Mr. Niinistö’s performance was quite lacklustre (eg. Hallamaa, 2012; Iranto, 2012). It could be asked whether his position as the predicted winner meant that he did not see
the need for campaigning. Second, the 2012 presidential elections were the first elections in Finland with an openly
homosexual candidate: Mr. Pekka Haavisto from the Green Party, who eventually proceeded to the second round of the
elections with Mr. Niinistö. According to estimates it is clear Haavisto’s sexual preference was a central factor with the
elections, with many people choosing to vote for his rival because they were not ready to have an openly homosexual
president in the country (Blencowe, 2012). Similarly, many people rallied to vote for Haavisto because they wanted to
show support for gay rights in Finland. 5. Context of the 2012 Finnish Debates Finland is a parliamentary democracy, in which parliament is formed by multiple parties and governed by the Prime
Minister. Unlike in a presidential system, in Finland the president has relatively little political power; the tasks of the
president have been reduced several times, the latest of which took place in 2000 and left the president with little
political power mainly consisting of international relations. However, even in that field the tasks of the president are
restricted: for example matters concerning the European Union are mainly handled by the Prime Minister (Halonen,
2002). It has been claimed that these days the role of the president in Finland is mostly symbolic, that of representing
the nation and its values to the outside world (Halonen, 2002). Nevertheless, there are still those in Finland who long for
a strong president to lead the country and its politicians (Halonen, 2002). The president is elected every six years and can have two consecutive terms of office. The president is elected through a
direct vote. Since Finland is a multi-party system, there are always several candidates. Should one of these candidates
get more than 50 percent of the votes in the first round, that candidate is elected president. If none of the candidates get
more than half of the votes, the elections proceed to a second round, in which the president is elected among the two
candidates who gained the most votes in the first round. Usually the second round is needed before a winner can be
determined. Even though the president has relatively little political power, presidential elections matter: ever since the
late 1980’s, the voter turnout at presidential elections has been about 10 percent higher than in parliamentary elections
(Moring, 2008, in Isotalus, 2011). One possible reason is the people still perceive the president as their leader. Another
reason might be the fact that voting for president is considered to be “easier” than voting in the parliamentary elections
as there are fewer candidates to choose from. In the 2012 presidential elections in Finland there were originally eight candidates, none of whom was the incumbent as
President Tarja Halonen was leaving the office after two consecutive terms. A clear favourite according to the polls was
Mr. Sauli Niinistö from the National Coalition Party (Yle, 2012). 4. Method In the
Finnish presidential debates, however, the role of the moderators is very visible. Their questions are guiding the 73 Studies in Media and Communication Vol. 4, No. 2; 2016 discussions, and they are even actively making attacks against the candidates. Since these attacks frequently forced the
candidates to defend themselves, it was decided they should be included in the analysis. All the other utterances made
by the moderators were left unanalysed. discussions, and they are even actively making attacks against the candidates. Since these attacks frequently forced the
candidates to defend themselves, it was decided they should be included in the analysis. All the other utterances made
by the moderators were left unanalysed. 5. Context of the 2012 Finnish Debates This challenges the assumption made by the functional theory of political
campaign discourse that policy matters more than character: clearly, in the 2012 presidential elections in Finland,
personal characteristics, not political expertise, were a decisive factor (Blencowe, 2012). 6. Results A total of 331 turns were coded. These included all of the turns of the candidates (Niinistö, 153 turns, Haavisto, 144
turns) as well as attacks uttered by moderators (34 turns). Most of these turns could be categorised into functions;
however, 91 of the turns (28%) could not be categorized into these categories. Each utterance was classified as policy or
character. Policy comments were further divided into past deeds, future plans, and general goals. Character themes were
subdivided into personal qualities, leadership ability, and ideals. Hypothesis 1 predicted acclaims would be the most used function in the Finnish presidential debates, followed by 74 Vol. 4, No. 2; 2016 Studies in Media and Communication attacks and finally, defences. In the analysed debates, there was a significant difference between the different functions:
χ2 (df = 2) = 98.29, p < .0001. Overall, acclaims were the most preferred statement by the candidates (n = 120), with
agreement being the least preferred (n = 15). The results can be seen in Table 1. In the first debate, Niinistö made an
acclaim concerning his character and personal qualities: attacks and finally, defences. In the analysed debates, there was a significant difference between the different functions:
χ2 (df = 2) = 98.29, p < .0001. Overall, acclaims were the most preferred statement by the candidates (n = 120), with
agreement being the least preferred (n = 15). The results can be seen in Table 1. In the first debate, Niinistö made an
acclaim concerning his character and personal qualities: My reason for participating in these elections from the very beginning has been the strong knowledge that I have the
capability of dedicating myself to what I’m doing at any specific moment, and I believe I have lots to give when it
to making sure that good life will exist in Finland also in the future. That is the goal I want to serve and I dedicate
to that task. However, it should be taken into account that most of the attacks recorded were actually made by moderators (67%) –
should the attacks made by moderators be left out, the results would be different, with acclaims being the most used
function, defences the second and attacks the third. 6. Results This was supported: with a significant difference
between the topics: χ2 (df = 1) = 90.30, p < .0001. As predicted, policy (n = 142) was discussed more than character (n
76). 22 turns (30%) could not be classified into either policy or character, corresponding with the percentage of turns
not categorised into functions. 44% of Niinistö’s utterances concerned policy and 19% concerned character (37%
uncategorised), while Haavisto discussed policy in 54% of his turns and character in 15% of the turns (31%
uncategorised). The results of this can be seen in Table 2. Both policy and character were also topics of attacks made by
moderators: policy was used in 14% of the moderators’ attacks, while character was the topic of 69% percent of these
attacks. The rest of the attacks could not be assigned into either of these topic categories. One of the moderators
provided an example of a character-focused attack: Hypothesis 2 predicted policy would be discussed more than character. This was supported: with a significant difference
between the topics: χ2 (df = 1) = 90.30, p < .0001. As predicted, policy (n = 142) was discussed more than character (n
76). 22 turns (30%) could not be classified into either policy or character, corresponding with the percentage of turns
not categorised into functions. 44% of Niinistö’s utterances concerned policy and 19% concerned character (37%
uncategorised), while Haavisto discussed policy in 54% of his turns and character in 15% of the turns (31%
uncategorised). The results of this can be seen in Table 2. Both policy and character were also topics of attacks made by
moderators: policy was used in 14% of the moderators’ attacks, while character was the topic of 69% percent of these
attacks. The rest of the attacks could not be assigned into either of these topic categories. One of the moderators
provided an example of a character-focused attack: Last Sunday at the election results party you said that everyone needs someone in their house who cooks for them,
their shirts and takes care of them. Now you’ve had to many give explanations for this statement. Did you accidentally
happen to reveal something real about your attitude, Sauli Niinistö? Table 2. 6. Results In the following passage, one of the moderators attacked Haavisto: It is said about you, Pekka Haavisto, that you know people from the Russian opposition and non-governmental
organisations, but you do not have any ties to the Russian leadership in Kreml. Just what kind of president would you
when you do not even have any ties to Kreml? The attacks made by moderators were often very direct, while the attacks made by the candidates themselves were not
so direct. The candidates would for example disagree with facts presented by the other candidate, or question their
abilities in some other way. In the following passage, Haavisto attacked Niinistö about the funding of his campaign: The attacks made by moderators were often very direct, while the attacks made by the candidates themselves were not
so direct. The candidates would for example disagree with facts presented by the other candidate, or question their
abilities in some other way. In the following passage, Haavisto attacked Niinistö about the funding of his campaign: Well dependability is, I’m not accusing you Sauli about anything, but the fact is that when one has a lot of big
it does bring into mind the question that what is the interest of these large companies, and president’s trade promoting
functions and so on. Even though Haavisto claimed he is not making any accusations, the paragraph above is clearly an attack questioning
Niinistö’s integrity and financial dependability. Finally, Isotalus (2011) suggested that agreements are an important
function of Finnish presidential debates. In the debates analysed, agreements formed 5% of all the turns, indicating they
are used to some extent, but other functions are still much more common. Table 1. Functions of the 2012 Finnish presidential debates
Acclaims
Attacks
Defences
Agreements
Niinistö
53 (35%)
10 (7%)
29 (19%)
8 (5%)
Haavisto
68 (47%)
7 (5%)
22 (15%)
7 (5%)
Moderators
-
35 (67%)
-
-
Total
120 (36%)
52 (16%)
51 (15%)
15 (5%) Table 1. Functions of the 2012 Finnish presidential debates Table 1. Functions of the 2012 Finnish presidential debates
Acclaims
Attacks
Defences
Agreements
Niinistö
53 (35%)
10 (7%)
29 (19%)
8 (5%)
Haavisto
68 (47%)
7 (5%)
22 (15%)
7 (5%)
Moderators
-
35 (67%)
-
-
Total
120 (36%)
52 (16%)
51 (15%)
15 (5%)
Hypothesis 2 predicted policy would be discussed more than character. 6. Results Topics of 2012 Finnish presidential debates
Policy
Character
Niinistö
67 (44%)
29 (19%)
Haavisto
78 (54%)
22 (15%)
Moderators
6
18
Total
151 (46%)
79 (24%)
Hypothesis 3 predicted general goals would be used more frequently to acclaim than to attack in the 2012 Finnish
general presidential debates. This hypothesis was supported: χ2 (df = 1) = 15.16, p < .05. As predicted, general goals (n
= 131) were used more frequently to acclaim (n = 94) than to attack (n = 13). These results can be seen in Table 3. Hypothesis 3 predicted general goals would be used more frequently to acclaim than to attack in the 2012 Finnish
general presidential debates. This hypothesis was supported: χ2 (df = 1) = 15.16, p < .05. As predicted, general goals (n
= 131) were used more frequently to acclaim (n = 94) than to attack (n = 13). These results can be seen in Table 3. Hypothesis 3 predicted general goals would be used more frequently to acclaim than to attack in the 2012 Finnish
general presidential debates. This hypothesis was supported: χ2 (df = 1) = 15.16, p < .05. As predicted, general goals (n
= 131) were used more frequently to acclaim (n = 94) than to attack (n = 13). These results can be seen in Table 3. 75 Vol. 4, No. 2; 2016 Studies in Media and Communication Table 3. Subtopics of the Finnish 2012 presidential debates
Functions
Acclaims
Attacks
Defences
Agreements
Subtopic
Past deeds
5 (5%)
2 (13%)
3 (20%)
0 (0%)
General Goals
94 (95%)
13 (87%)
11 (73%)
13 (100%)
Not categorised
0 (0%)
0 (0%)
1 (7%)
0 (0%)
Total
99
15
15
13
Hypothesis 4 predicted ideals would be used more frequently to acclaim than to attack in the 2012 Finnish general
presidential debates. This hypothesis was supported: χ2 (6) = 16.28, p < = .05. As predicted, ideals (n = 14) were used
more frequently to acclaim (n = 8) than to attack (n = 3). These results can be seen in Table 4. Table 3. Subtopics of the Finnish 2012 presidential debates Table 4. 6. Results Use of ideals in the Finnish 2012 presidential debates
Functions
Acclaims
Attacks
Defences
Subtopics 2
Leadership ability
6 (29%)
7 (26%)
9 (30%)
Personal qualities
5 (24%)
17 (63%)
18 (60%)
Ideals
8 (38%)
3 (11%)
3 (10%)
Not categorised
2 (10%)
0 (0%)
0 (0%)
Total
21
27
30 Table 4. Use of ideals in the Finnish 2012 presidential debates Table 4. Use of ideals in the Finnish 2012 presidential debates Coding the turns to different functions was challenging. Although categorising themes into defence and agreements was Coding the turns to different functions was challenging. Although categorising themes into defence and agreements was
fairly easy, a broader approach had to be taken with acclaims and attacks. In Finnish debates, candidates rarely made
clear statements declaring a certain course of action they would take up as a president (such as “I will cut the taxes” or
“I will decrease unemployment”). Instead, they expressed their opinion on policies they generally perceived as desirable
or on the direction they would like to see the country to go in the future. In the context of this study, these were
nevertheless classified as acclaims, as they were understood to be policies the candidates would drive forward should
they have the opportunity. The same perspective was applied to attacks: even utterances that were not direct attacks
would be classified into that category if they contained a clear criticism or challenge towards the other candidate. A notable amount of turns could not be classified into any category. These included, among others, jokes made by the
candidates, reacting to Twitter comments made by audience members, as well as analysing the current political situation
in globally or in Finland. For example: I think we need to be very careful when it comes to these terms. Binding ourselves to the West is too broad a term, it
includes two different elements. Since we joined the EU this old term called neutrality is no longer so relevant,
because as EU members we do express our opinions, we express our opinions on the crisis in Libya, we express our
opinions on Iran and so on. Although statements such as these cannot be categorised as acclaims, they nevertheless do have a role in painting a
picture of the candidate as an expert on national and global politics, which might cause voters to see them in a more
favourable light. 6. Results In future studies it might be interesting to add another function – expressions of expertise – to research
these turns further. 7. Discussion Also all
significant decisions in the area of foreign policy made by the president must be done in accordance with the
government. With president’s power in Finland being mostly symbolic, it could be questioned why so much of the
discussion still happens on the level of policy. The president’s diminished duties and power are also likely visible in the way policy is discussed in these debates. An
overwhelming amount (85%) of all policy utterances were made on the level of general goals; only 6% of the utterances
focused on past deeds, and future plans were not discussed at all. While the difference between general goals and future
plans also exists in the US debates, it is not as drastic as in the Finnish debates. In the United States it is still possible to
see candidates making promises to cut taxes or to increase the military spending, yet in the Finnish debates analysed,
this did not happen. The most likely reason is the president’s limited power in Finland: there is little point for the
candidates to present elaborate future plans for their turn as a president when they do not possess the political power to
make those plans into reality. Instead, it makes much more sense to discuss on the level of general goals – policies that
the candidates see as generally desirable, at the same time acknowledging that they might not be able to act on those
policies. This was again demonstrated by Niinistö in one of the debates where he first outlines his view of the economic
situation in Finland and the direction he would like to see it go and then reminds the audience that the president does
not have the power to decide on matters of economics in Finland. Perhaps the clearest difference between the presidential debates in the US and in Finland is the number of themes that
did not fit into any category. In the presidential debates in the US the percentage of themes left uncoded is very small; in
the debates analysed here, the portion is larger (28%). Isotalus (2011) produced similar results. Isotalus (2010, 2011)
claimed that agreements form a large portion of the themes left uncoded. 7. Discussion The results of this analysis are generally consistent with the results of previous Functional Theory studies of presidential
elections in the United States and other countries: Candidates used acclaims more than attacks or defences and policy
was discussed more than character. However, some differences did emerge. Most of the attacks were uttered by the
moderators, meaning that the candidates themselves used more defences than attacks. It is possible the reason for this
difference lies in the different formats of the presidential debates; in the Finnish debates, the moderators are clearly in
charge of the discussion, asking questions and making attacks against the candidates. This situation forces the
candidates to react to the questions and attacks posed by the moderators and leaves them with relatively little room to
engage in a direct discussion with one another - thus they simply do not have the time to attack each other. Another
reason might be the fact that Niinistö was predicted as the clear winner throughout the whole campaign – perhaps the
candidates did not see any point in making attacks, when the results seemed to be already decided. This is even hinted at
by Niinistö in one of the debates when he, accused by one of the moderators as having been more quiet than normal,
states that he sees little point in fighting with the others for the second place, when the results are already clear. Policy was discussed more than character in the Finnish debates. While this result again correlates with the results from
the US, it is slightly contradictory with statements from election experts, in which they claim that character, in fact, was
the decisive factor in the 2012 election: namely the facts that Haavisto is homosexual, does not belong to any church
and never served in the army, but opted for civil service instead (Blencowe, 2012). These are all questions of personal
values, and while they were discussed shortly in the debates, much more time was dedicated for discussion of policy. 76 Vol. 4, No. 2; 2016 Studies in Media and Communication This is also interesting considering the fact that in Finland, president has little say over any actual policies. President has,
for example, the duty to confirm Acts into laws, but Acts can be entered into force even without the president’s
confirmation - leaving legislative power essentially in the hands of the government and the parliament. 7. Discussion However, the analysis here shows that the
portion of agreements is not all that significant: only 5% of all the turns coded, meaning that even with agreements, the
percentage of uncoded turns would not be more than 33%. Therefore, the majority of the uncoded turns still consist of
something else besides agreements. To some extent, these turns are “empty speech” – jokes, reactions to comments
from audience et cetera. However – as has been noted by Isotalus (2010, 2011) earlier, these turns also include
something that could perhaps best be characterised as “analysis of the current political / economic / societal situation in
Finland/globally.” The candidates not only made these analysing statements themselves, but also questioned the
accuracy of the other person’s analysis or facts. In this sense it could be argued that these analyses serve a purpose in
political campaign discourse: the candidates attempt to paint a picture of themselves as experts as well as question the
expertise of the other candidate(s), in a way attempting to claim to have the right narrative on how the world works. While these expressions of expertise have not been reported to appear in the US presidential debates, it is possible that
they are a meaningful part of Finnish political campaign discourse and should be paid attention to in the future. 8 C
l
i 8. Conclusions This study indicates there are both similarities as well as differences between the political campaign discourse of
presidential debates in Finland and the US. However, neither earlier research nor the scope of the present study provides
sufficient information on the significance of these differences: more research on the Finnish presidential debates would
have to be conducted to determine whether the differences exist in all debates or whether they are simply a part of this
particular campaign. The 2012 campaign was special for many reasons: the predicted clear victory for Mr Niinistö, the
existence of an openly homosexual candidate and the historically low voter turnouts. Based on the special nature of the
campaign, it would be presumptuous to assume that the results could be generalised to all presidential campaigns in
Finland. In this research, debates from the second round of the elections were chosen because their format resembled more
closely the format of the US presidential debates. In the future, it could be interesting to analyse the debates from the
first round of elections, with all the eight candidates present, and see whether there is any difference in the results. Another interesting possibility would be to study debates from the second round of 2012 elections, broadcast by
different commercial broadcasting companies. These debates by different broadcasting companies might also have
different formats, which could possibly be helpful in trying to determine the extent to which these differences are
caused by formats, and to which extent they are caused by actual cultural differences. This would also help to determine
whether the functional theory of political campaign discourse is a suitable tool for analysing Finnish presidential
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http://yle.fi/uutiset/suomen_seuraava_presidentti_on_joko_konservatiivi_tai_liberaali/5053272 Schrott, P. R. (1990). Electoral consequences of “Winning” televised campaign debates. Public Opinion Quarterly, 54,
567-585. http://dx.doi.org/10.1086/269228 Straub, S. K., Beller, J. L., & Hunt, T. W. (2012). I concur, you are absolutely correct that I am correct: Agreement as an
argumentative strategy. Contemporary Argumentation & Debate, 33, 1-26. Tiittula L., Nuolijärvi, P., & Isotalus, P. (2007). Halosen ja Niinistön esiintymistyylit television vaalikeskusteluissa. In
Isotalus, P. & Borg, S. (Eds), Presidentinvaalit 2006. (pp. 155-177). Helsinki, Finland: WSOY. References Lanham, MD: University Press of
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argumentoivat presidentinvaalien 2006 vaalitenteissä. Master’s Thesis. The University of Jyväskylä: Jyväskylä. Kinnunen, P. (3 August, 2011). Kuka haastaisi Niinistön? Yle Uutiset. Retrieved from:
http://yle.fi/uutiset/kuka_haastaisi_niiniston/5401052 Kinnunen, P. (3 August, 2011). Kuka haastaisi Niinistön? Yle Uutiset. Retrieved from:
http://yle.fi/uutiset/kuka_haastaisi_niiniston/5401052 Kuivasmäki, A. (2000). Martti Ahtisaaren ja Elisabeth Rehnin viestintätyyli television vaaliohjelmissa vuoden 1994
presidentinvaaleissa. Master’s Thesis. The University of Jyväskylä: Jyväskylä, Finland. Lee, C., & Benoit, W. L. (2004). A functional analysis of Presidential television spots: A comparison of Korean and
American ads. Communication Quarterly, 52, 68-79. http://dx.doi.org/10.1080/01463370409370179 Lee, C., & Benoit, W. L. (2005). A functional analysis of the 2002 Korean Presidential debates. Asian Journal of
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effects. Argumentation & Advocacy, 49, 238-258. Peifer, J. T., & Holbert, R. L. (2013). Developing a systematic assessment of humor in the context of the 2012 U.S. general election debates. Argumentation & Advocacy, 49, 286-300. fau, M. (2002). The subtle nature of presidential debate influence. Argumentation & Advocacy, 38, 25 Rhea, D. (2012). There they go again: The use of humor in Presidential debates 1960-2008. Argumentation & Advocacy,
49, 115-131. Roitman, M. (2015). Constructing one’s arguments based on refutations of the other’s discourse. A study of the
traditional Presidential debate: Chirac/Jospin (1995) versus Sarkozy/Royal (2007). Argumentation, 29, 19-32. http://dx.doi.org/10.1007/s10503-014-9332-4 79 Studies in Media and Communication Vol. 4, No. 2; 2016 Schrott, P. R. (1990). Electoral consequences of “Winning” televised campaign debates. Public Opinion Quarterly, 54,
567-585. http://dx.doi.org/10.1086/269228 Straub, S. K., Beller, J. L., & Hunt, T. W. (2012). I concur, you are absolutely correct that I am correct: Agreement as an
argumentative strategy. Contemporary Argumentation & Debate, 33, 1-26. Tiittula L., Nuolijärvi, P., & Isotalus, P. (2007). Halosen ja Niinistön esiintymistyylit television vaalikeskusteluissa. In
Isotalus, P. & Borg, S. (Eds), Presidentinvaalit 2006. (pp. 155-177). Helsinki, Finland: WSOY. YLE. (23 January, 2012). Suomen seuraava presidentti on joko konservatiivi tai liberaali. Yle Uutiset. References Retrieved from:
http://yle.fi/uutiset/suomen_seuraava_presidentti_on_joko_konservatiivi_tai_liberaali/5053272 Zarefsky,
D. (2008). Strategic
maneuvering
in
political
argumentation. Argumentation,
22,
317-330. http://dx.doi.org/10.1007/s10503-008-9096-9 This work is licensed under a Creative Commons Attribution 3.0 License. This work is licensed under a Creative Commons Attribution 3.0 License 80
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Surgical care in the public health agenda
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Surgical care in the public health agenda Mário Scheffer 1
Saurabh Saluja 2,3
Nivaldo Alonso 1 doi: 10.1590/0102-311X00104717 1 Faculdade de Medicina, Universidade de São Paulo, São Paulo,
Brasil.
2 Program in Global Surgery and Social Change, Harvard
Medical School, Boston, U.S.A.
3 Department of Surgery, Weill Cornell Medical College,
New York, U.S.A. This article is published in Open Access under the Creative Commons
Attribution license, which allows use, distribution, and reproduction in
any medium, without restrictions, as long as the original work is correctly
cited. ESPAÇO TEMÁTICO: CUIDADOS CIRÚRGICOS E SAÚDE PÚBLICA
THEMATIC SECTION: SURGICAL CARE AND PUBLIC HEALTH Cad. Saúde Pública 2017; 33(10):e00104717 Abstract Correspondence
M. Scheffer
Faculdade de Medicina, Universidade de São Paulo. Av. Dr. Arnaldo 455, sala 2221, São Paulo, SP
01246-903, Brasil. mscheffer@uol.com.br The current article examines surgical care as a public health issue and a chal-
lenge for health systems organization. When surgery fails to take place in
timely fashion, treatable clinical conditions can evolve to disability and death. The Lancet Commission on Global Surgery defined indicators for monitoring
sustainable universal access to surgical care. Applied to Brazil, the global in-
dicators are satisfactory, but the supply of surgeries in the country is marked
by regional and socioeconomic inequalities, as well as between the public and
private healthcare sectors. 1 Faculdade de Medicina, Universidade de São Paulo, São Paulo,
Brasil. 2 Program in Global Surgery and Social Change, Harvard
Medical School, Boston, U.S.A. 3 Department of Surgery, Weill Cornell Medical College,
New York, U.S.A. Operative Surgical Procedures; Global Health; Health Systems Cad. Saúde Pública 2017; 33(10):e00104717 Scheffer M et al. Access to surgical care has been neglected and unrecognized as a public health problem 1. Recently,
however, it has been incorporated into the global health research agenda, resulting in a growing
knowledge base of how surgery can be incorporated into health policy 2,3. The Lancet Commission on Global Surgery published a report in 2015 that frames the provision
of surgical care as an essential part of health systems. The report advocates for the prioritization of
surgery and the integration of principles of universality, equity, and justice into surgical systems. Furthermore, the report emphasized the economic argument that surgical care is cost-effective, even
in areas of limited resources 2. Scientific advances in surgical care – which include both an evolution in procedures and incor-
poration of new technologies – have improved the diagnosis and treatment of health problems. Yet,
these advances have not been shared across different health systems, regions, or socioeconomic
groups. In many countries and localities, access to surgical care is limited by the scarcity of surgeons,
anesthetists and obstetricians 4,5. Nearly 5 billion people worldwide do not have access to safe, afford-
able, and timely surgical care, and only 6% of total surgical procedures occur in the world’s poorest
countries. Moreover, it is estimated that 16.9 million lives were lost in 2010 due to health problems
that required surgical care 6. Abstract Surgical disease manifests across all age-groups and affects the care
of many conditions that are prioritized in public health. It plays a critical role in trauma, maternal
and child health, and cancer; ultimately, surgery is estimated to play a role in 28-32% of the global
burden of disease 7. The unavailability of timely surgical care can transform treatable conditions into diseases with
high mortality and morbidity, besides adversely affecting the economy of countries and the well-
being of populations 8. For laboring women, unresolved surgical complication can lead to the death
of mother and child; congenital malformations, such as cleft lip, and acquired conditions, such as
burns, if left uncorrected, can harm the cognitive and social development of individuals; and delays
in trauma care and fractures can leave young people disabled and economically unproductive for life. The surgical system is ample and includes professionals, institutions, as well as public and private
financial sectors responsible for service delivery. The surgical system is involved across the spectrum
of disease, ranging from prevention to diagnostics to treatment and palliation. It requires human
resources, consumables and equipment, which are used in the preoperative evaluation of patients’
health and risk factors, the intraoperative period during which procedure occurs, and the postopera-
tive period. To this end, the surgical system requires infrastructure consisting of surgical facilities,
blood supply, referral systems as well as human resources capable of anesthetic, surgical and obstetric
care. This includes physicians, surgical teams, nursing personnel, and technicians responsible for
radiology, pathology, and laboratory. The lack of attention to surgical care in health-systems and health policy research agendas
may be linked to the mistaken notion that surgical care applies to only a small portion of diseases,
that it is prohibitively complex, or not cost-effective. It may also be the consequence of choices
by governments that favor healthcare models that neglect the role of surgery in health-systems. The result is policies and funding for public health research that neglect surgical care, especially in
developing countries. While surgical care is often cost-effective at the system level, at the individual level it often
includes costly procedures. It is responsible for much of the remuneration of doctors and hospitals in
both public and private systems and it interfaces with the medical industry, including pharmaceutical
companies and equipment and supply companies. Cad. Saúde Pública 2017; 33(10):e00104717 Abstract The provision of surgical care is therefore sensitive
to individual health demands, physician decisions, and private business interests. It must also be the
object of collective management – i.e. it must be structured by health systems. The role of the state is,
therefore, fundamental to ensure the delivery and function of surgical services and the availability of
human resources. Part of that role includes regulation and standardization of care. It has been shown that patients
with similar health problems that require the same surgical intervention are treated differently from
one country to another and even within the same country 9,10. For example, the number of patients
admitted for surgery is twice as high in Germany, Australia, and Israel than in Canada, Spain, and
Portugal. A patient is three times more susceptible to have a cardiac revascularization in Germany
and Israel when compared with other countries. In France 11, an analysis of ten surgical interventions Cad. Saúde Pública 2017; 33(10):e00104717 SURGICAL CARE IN THE PUBLIC HEALTH AGENDA
3 showed persistent variation in medical practice, both at the regional level and within departments. This applied to both the frequency and effectiveness of interventions. Barriers to access, variations in practice patterns and unavailability of care create ethical, thera-
peutic, and economic problems for the health system. To understand the scale of these problems, more
studies using public information on the availability of surgical services, their geographic variation, as
well as different practice patterns are needed. In 2015, the Lancet Commission on Global Surgery selected six indicators and a framework for a
national surgical plan to help evaluate the current state of surgical and anesthesia systems. The reason
to define these indicators was to allow each country to measure its current state of surgical service
provision and, with this information, form a national surgical plan. The indicators presented by the
commission include timely access to essential surgical care, adequacy of surgical workforce, volume
of surgical procedures, post-operative mortality rate, and protection against impoverishing expendi-
tures owing to surgical care. Recently applied to Brazil, these indicators proved to be useful and revealed a country with gen-
erally adequate parameters but with immense geographic inequality and regional differences in the
ability to offer surgery and anesthesia. Abstract With a workforce density (defined as surgeons, anesthetists
and obstetricians) of 34.7 per 100,000 habitants, a public surgical volume of 4,433 surgical procedures
per 100,000 habitants, and post-operative mortality rate of 1.71%, the country achieved indicators
close to suggested values. However, there are regional disparities in each of the suggested indicators,
with some regions and states having indicators close to those of low-income countries 12. The density
of the surgical workforce varies from 20.55 per 100,000 people in the North up to 61.94 per 100,000
people in the South. Overall, anesthetists make up 19.8% of the surgical workforce, which is right at the
worldwide mean of 20%. However, in the states of Rondônia, Roraima, Amapá, Maranhão and Piauí,
anesthesiologists make up around 14% or less of the surgical workforce 13. Brazil has a surgical work-
force that easily exceeds the suggested benchmark of 20 surgical specialists per 100,000 habitants. However, more than 70% of these professionals were in large cities where only 24% of the population
lives. Moreover, these professionals are largely concentrated in private health institutions, which only
serve the quarter of the Brazilian population that has private health insurance. In December 2016, a meeting in São Paulo addressed surgical care provision and assessed the
viability of implementing national surgical plans throughout Latin America. At this meeting, a pilot
study that analyzed surgical system in the state of Amazonas was presented 14. The study used quali-
tative methods to interview surgeons, anesthetists, and other health professionals from hospitals in
the interior, aiming to understand the Lancet Commission on Global Surgery indicators and access
emergency surgeries, such as cesarean section, open fracture, and laparotomy. These interviews
revealed a severe lack of health professionals, resulting in non-surgeons providing surgical care, and
non-physicians performing medical procedures. Many interviewees addressed poor work conditions,
quality of life, and remuneration as reasons for the unavailability of health professionals. The availability of surgical services, or lack therein, is an inseparable part of the Brazilian health
system that is susceptible to the financing, function, and organization of the system as a whole. Access
to healthcare is not simply related to the availability of health services, but is also influenced by social
determinants of individuals and by their locale of residence 15. Abstract The wait-time to receive specialized
medical care and elective surgery is shorter for people with higher incomes and longer for those of
lower socioeconomic status, even after adjusting for age, sex, education, and state of health. Further,
when evaluating hospitalizations in Brazil, some of which are for surgical procedures, there has been a
trend for many years (from 1998 to 2013) towards decreasing direct payments from patients, decreas-
ing hospitalizations attributed to the Brazilian Unified National Health System (SUS), and increasing
hospitalizations for private health plans 16. The SUS, which provides free at point of service universal healthcare, prevents and treats diseases,
besides having a complex mix of directly and indirect administration by the federal government,
states, and municipalities. It is organized in a regionalized and hierarchical form and relies on comple-
mentary private initiatives towards the provision or management of healthcare 17. Approximately one in four Brazilians has private health insurance, which, in many cases, covers
surgery. However, this population also uses surgical services offered by SUS, whether in prevention
campaigns, urgent or emergent situations such as motor vehicle accidents, for surgeries not covered Cad. Saúde Pública 2017; 33(10):e00104717 Scheffer M et al. 4 by private health plans, or in the case of cancelled insurance due to unemployment (more than 80% of
the insurance market is comprised of plans offered by employers). While most primary care and emer-
gency services are public, hospitals, diagnostic services, and radiology are mostly private, and private
expenditure represents approximately 54% of total healthcare expenditure in Brazil 18. This result is
a concentration of resources in a small private sector that selects against high-risk patients and pro-
cedures and an under-resourced public sector tasked with the comprehensive care of the population. This manifests a distortion in the provision of services. For example, the public availability of mid-
complexity care, which includes ambulatory surgery, is insufficient in Brazil. These services are most-
ly offered in the private sector and primarily serve patients with health plans that reimburse more
than SUS does for surgical procedures. Whether in free-standing facilities, adjacent to specialty clin-
ics, or in hospitals with surgical centers, ambulatory surgery is characterized by same-day discharge. Ambulatory surgery can be used in varied situations, such as cataract operations, knee arthroscopy,
varicose vein surgery, etc. Abstract It has the benefit of using fewer resources, reducing cost, decreasing wait-
lists, improving patient-satisfaction, and limiting exposure to nosocomial infections – outcomes that
should guide the expansion of such services in the public health system. While ambulatory surgery represents a potential for improving the delivery of public surgical
services in Brazil, the hospital network remains the backbone of surgical care delivery. However,
this network is considered heterogeneous and inefficient and relies on a predominance of small to
mid-sized hospitals (up to 50 beds), with outdated infrastructure, that provide inadequate service to
the population 19. There are disparities among regions and between public and private sectors, with
the paradoxical result of having idle beds in some hospitals whilst there is overflow in others. This is
driven, in part, by differential pricing for the same type of hospitalization or procedure. In Brazil, only
35% of hospital beds are in public hospitals with the remaining 65% in the private sector, with many of
these hospital networks organized into private financial groups. Since the public availability is insuffi-
cient, SUS must purchase from the private sector. Thus, providers and purchasers of surgical services
compete, and governments and health plans pay differential rates for hospitalizations and surgeries. Surgeons and anesthetists have dual practices, but often concentrate their time in the private sector
due to better work conditions and remuneration. The result is an exacerbated inequality in utilization
of surgical services, especially for elective care. The future of surgical care in Brazil depends on the sustainability of SUS, which, in turn, depends
on overcoming political and economic crises that have afflicted the country since 2013. These crises
have resulted in fiscal adjustments, diminishing resources for public health and the emergence of
proposals to expand the private health market by developing “accessible” or “popular” plans 20, offer
reduced coverage, and may not cover hospitalizations or surgeries. While this may be an appealing
approach to austerity, it may paradoxically result in longer surgical wait-times and continued over-
burdening of SUS, especially for surgical conditions. Surgical care without a doubt must be incorporated into all levels of the health system, and coun-
tries must integrate safety, quality and cost-effectiveness in the provision of services. To do so, data
collection systems are needed to evaluate and monitor clinical processes, costs, and outcomes. References The authors contributed equally in the production
of the paper. 1. Farmer PE, Kim JY. Surgery and global health:
a view from beyond the OR. World J Surg
2008; 32:533-6. 2. Meara JG, Leather AJ, Hagander L, Alkire
BC, Alonso N, Ameh EA, et al. Global surgery
2030: evidence and solutions for achieving
health, welfare, and economic development. Lancet 2015; 386:569-624. 3. Dare AJ, Grimes CE, Gillies R, Greenberg SL,
Hagander L, Meara JG, et al. Global surgery:
defining an emerging global health field. Lan-
cet 2014; 384:2245-7. 4. Holmer H, Lantz A, Kunjumen T, Finlayson S,
Hoyler M, Siyam A, et al. Global distribution
of surgeons, anaesthesiologists, and obstetri-
cians. Lancet Glob Health 2015; 3 Suppl 2:
S9-11. 5. Lantz A, Holmer H, Finlayson S, Ricketts TC,
Watters D, Gruen R, et al. International migra-
tion of surgeons, anaesthesiologists, and ob-
stetricians. Lancet Glob Health 2015; 3 Suppl
2:S11-2. 6. Weiser TG, Regenbogen SE, Thompson KD,
Haynes AB, Lipsitz SR, Berry WR, et al. An
estimation of the global volume of surgery:
a modelling strategy based on available data. Lancet 2008; 372:139-44. 7. Shrime MG, Bickler SW, Alkire BC, Mock C. Global burden of surgical disease: an estima-
tion from the provider perspective. Lancet
Glob Health 2015; 3 Suppl 2:S8-9. 8. Murray CJ, Vos T, Lozano R, Naghavi M, Flax-
man AD, Michaud C, et al. Disability-adjusted
life years (DALYs) for 291 diseases and injuries
in 21 regions, 1990-2010: a systematic analysis
for the Global Burden of Disease Study 2010. Lancet 2012; 380:2197-223. 9. Organisation for Economic Co-operation and
Development. Geographic variations in health
care: what do we know and what can be done
to improve health system performance? Paris:
OECD Publishing; 2014. (OECD Health Policy
Studies). 10. Corallo AN, Croxford R, Goodman DC, Bryan
EL, Srivastava D, Stukel TA. A systematic re-
view of medical practice variation in OECD
countries. Health Policy 2014; 114:5-14. 11. Institut de Recherche et Documentation en
Économie de la Santé. Atlas des variations
de pratiques médicales recours à dix inter-
ventions chirurgicales. Paris: Institut de Re-
cherche et Documentation en Économie de la
Santé; 2016. 12. Massenburg BB, Saluja S, Jenny HE, Raykar
NP, Ng-Kamstra J, Guilhoux AGA, et al. As-
sessing the Brazilian surgical system with six
surgical indicators: a descriptive and model-
ling study. BMJ Glob Health 2017; 2:e000226. 13. Scheffer MC, Gilloux AGA, Matijasevich A,
Saluja S, Alonso N. Abstract These
systems will also shed light on the human and material resources needed for care delivery, the com-
plexity of public and private financing and management, and the way in which surgical services are
organized for, provided for, accessed by, and used by the population. The challenge of supporting a research agenda that brings surgical care closer to public health,
connects national and global problems, and finds ways to overcome inequalities to fully guarantee
the right to healthcare remains. Cad. Saúde Pública 2017; 33(10):e00104717 SURGICAL CARE IN THE PUBLIC HEALTH AGENDA
5 References The state of the surgi-
cal workforce in Brazil. Surgery 2017; 161:
556-61. Cad. Saúde Pública 2017; 33(10):e00104717 Scheffer M et al. 6 18. Paim J, Travassos C, Almeida C, Bahia L,
Macinko J. The Brazilian health system: his-
tory, advances, and challenges. Lancet 2011;
377:1778-97. 14. Saluja S, Citron I, Amundson J, dos Santos
Souza JE, Scheffer M, Ferreira RV, et al. Health
care leaders develop strategies for improving
acess to surgical care in Latin America. Bull
Am Coll Surg 2017; 102:22-7. 19. Brito LAL, Malik AM, Brito E, Bulgacov S,
Andreassi T. Práticas de gestão em hospitais
privados de médio porte em São Paulo, Brasil. Cad Saúde Pública 2017; 33:e00030715. g
15. Travassos C, Oliveira EXG, Viacava F. Desi-
gualdades geográficas e sociais no acesso aos
serviços de saúde no Brasil: 1998 e 2003. Ciênc
Saúde Coletiva 2006; 11:975-86. 20. Bahia L, Scheffer M, Dal Poz MR, Travassos
C. Private health plans with limited coverage:
the updated privatizing agenda in the context
of Brazil’s political and economic crisis. Cad
Saúde Pública 2016; 32:e00184516. 16. Viacava F, Bellido JG. Health, access to services
and sources of payment, according to house-
hold surveys. Ciênc Saúde Coletiva 2016;
21:351-70. 17. Secretaria de Gestão Estratégica e Participa-
tiva, Ministério da Saúde. Regulamentação da
Lei 8.080 para fortalecimento do Sistema Úni-
co da Saúde: decreto 7508, de 2011. Rev Saúde
Pública 2011; 45:1206-7. Cad. Saúde Pública 2017; 33(10):e00104717 Submitted on 19/Jun/2017
Approved on 28/Jun/2017 Resumen O presente artigo aborda os cuidados cirúrgicos
como um problema de saúde pública e um desafio
para a organização dos sistemas de saúde. Se ci-
rurgias não ocorrerem em tempo adequado, situa-
ções clínicas tratáveis podem evoluir para incapa-
cidades e mortes. A Lancet Commission on Global
Surgery definiu indicadores para o monitoramen-
to do acesso universal sustentável à assistência ci-
rúrgica. Aplicados ao Brasil, os indicadores globais
são satisfatórios, porém a oferta de cirurgias no
País é marcada por desigualdades entre regiões,
entre estratos socioeconômicos e entre os setores
público e privado da saúde. El presente artículo aborda la asistencia quirúrgi-
ca como un problema de salud público y un desafío
para la organización de los sistemas de salud. Si
no se ejecutan cirugías en el momento adecuado,
situaciones clínicas tratables pueden evolucionar
en incapacidades y muertes. La Lancet Commis-
sion on Global Surgery definió indicadores para
la supervisión del acceso universal sostenible a la
asistencia quirúrgica. Aplicados a Brasil, los in-
dicadores globales son satisfactorios, aunque la
oferta de cirugías en el país está marcada por des-
igualdades entre regiones, entre estratos socioeco-
nómicos y entre los sectores público y privado de
salud. Procedimentos Cirúrgicos Operatórios; Saúde
Global; Sistemas de Saúde Procedimientos Quirúrgicos Operativos; Salud
Global; Sistemas de Salud Submitted on 19/Jun/2017
Approved on 28/Jun/2017 Cad. Saúde Pública 2017; 33(10):e00104717
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Avaliando os impactos de políticas tributárias sobre a economia brasileira com base em um modelo de equilíbrio geral de gerações sobrepostas
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Revista Brasileira de Economia
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Avaliando os Impactos de Políticas
Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio
Geral de Gerações Sobrepostas Carlos Renato Salami∗, Adelar Fochezatto† 1. Introdução; 2. Estrutura do Modelo; 3. Simulações e Análise dos Resultados;
4. Comentários Finais. O objetivo deste trabalho é analisar os efeitos econômicos de longo
prazo de diferentes opções tributárias utilizando um modelo de equilíbrio
geral intertemporal com gerações sobrepostas. Considerando o aumento
do produto e do emprego como critério para identificar as melhores políti-
cas, os resultados permitem afirmar que: se o objetivo é reduzir a carga tri-
butária, a política recomendada é diminuir os impostos diretos; e se o obje-
tivo é substituir diferentes tipos de impostos, mantendo inalterada a carga
tributária, a recomendação é reduzir os impostos diretos e aumentar os in-
diretos. The objective of this study is to analyze the long-term economic effects
of different tax options using an intertemporal general equilibrium model with
overlapping generations. Considering the increase in output and employment
as a criterion to identify the best policies, the results show that: if the objective
is to reduce the tax burden, the recommended policy is to reduce direct taxes,
and if the objective is to replace various types of taxes, keeping unchanged the
tax burden, the recommendation is to reduce direct taxes and increase indirect
taxes ones. †Doutor em Economia. Professor Titular da PUCRS. Pesquisador do CNPq. Av. Neusa Brizola, 600/206, Bairro Petrópolis, CEP:
90460-230, Porto Alegre/RS. E-mail: adelar@pucrs.br ∗Mestre em Economia do Desenvolvimento. Professor da PUCRS. Rua Laurindo, 134/405, Bairro Santana, CEP 9 0040-140. Porto
Alegre/RS. E-mail: csalami@uol.com.br Alegre/RS. E-mail: csalami@uol.com.br 1. INTRODUÇÃO O sistema tributário atualmente em vigor no Brasil, implementado a partir da Reforma Constitu-
cional de 1988, foi concebido a fim de atenuar as distorções existentes na estrutura tributária herdada
do período militar. Isto foi feito por meio da reformulação na repartição das receitas públicas com vistas 299 RBE
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v. 63 n. 3 / p. 299–314
Jul-Set 2009 Carlos Renato Salami e Adelar Fochezatto à desconcentração dos recursos tributários da União em favor dos estados e municípios. Afonso et al. (2000) estima que a Reforma Constitucional tenha determinado uma perda financeira para a União da
ordem de 17% de suas receitas disponíveis e um aumento médio de 13,4% para os estados e de 31%
para os municípios. Além disso, os autores sustentam que a descentralização determinou perdas adi-
cionais em termos de eficiência do sistema porque ela provocou o surgimento de falhas de coordenação
entre as diferentes esferas de governo. à desconcentração dos recursos tributários da União em favor dos estados e municípios. Afonso et al. (2000) estima que a Reforma Constitucional tenha determinado uma perda financeira para a União da
ordem de 17% de suas receitas disponíveis e um aumento médio de 13,4% para os estados e de 31%
para os municípios. Além disso, os autores sustentam que a descentralização determinou perdas adi-
cionais em termos de eficiência do sistema porque ela provocou o surgimento de falhas de coordenação
entre as diferentes esferas de governo. A partir de então, tendo em vista a redução da receita sem a contrapartida de transferência de
encargos para os demais entes federados, o governo federal passou a agir no sentido de recompor as
suas perdas. Isto foi feito por meio da elevação das alíquotas das contribuições sociais existentes e
da criação de novas contribuições sociais. Isto porque o acréscimo de receita resultante dessas fontes
não necessitava ser dividido com os demais entes federados. Sendo assim, as elevações requeridas nas
alíquotas, para a obtenção de um dado acréscimo de receita, poderia ser menor do que a requerida,
por exemplo, nos casos do imposto sobre produtos industrializados ou do imposto de renda. Além
deste, outro esforço empreendido pela União foi a tentativa de transferir responsabilidades em relação
à prestação de serviços públicos para os demais níveis de governo. 1Trata-se, portanto, de um modelo do tipo Auerbach and Kotlikoff (1987). O modelo deste trabalho segue a estrutura apresentada
por Schubert and Letournel (1991). Em princípio, quanto maior o número de gerações, melhor. No entanto, um número muito
grande torna o modelo difícil de ser operado. As 11 gerações representam um meio termo e que reflete gerações de 5 anos cada. 1. INTRODUÇÃO As distorções provenientes da reforma tributária de 1988 levaram à elaboração, já nos anos seguintes
à sua promulgação, de muitas propostas de reformulação do sistema tributário. A última década foi
marcada por um contínuo debate sobre como proceder perante a reforma do sistema com o fim de se
eliminarem as suas distorções e aumentar a sua eficiência. Apesar disso, embora alguns avanços ten-
ham ocorrido, não se consolidou no período o entendimento necessário à concretização de mudanças
mais profundas. Dentre as principais distorções destacam-se a elevada regressividade, a excessiva com-
plexidade, o alto grau de descentralização e o excesso de carga tributária global. Inúmeros esforços têm sido realizados com o intuito de avaliar os impactos de curto e de longo prazo
das diferentes propostas de alterações no sistema tributário nacional. Entretanto, haja vista a complex-
idade do tema, os resultados apresentados não têm sido conclusivos a ponto de facilitar a tomada de
decisões. Isto talvez explique a dificuldade observada na obtenção de um consenso mínimo para a
aprovação de propostas mais profundas do atual sistema tributário brasileiro. A reforma aprovada em
2003 configura um exemplo claro desta situação. O objetivo deste trabalho consiste em realizar alguns exercícios envolvendo política tributária para
verificar os seus prováveis efeitos econômicos de longo prazo. Para isso, utiliza-se um modelo de equi-
líbrio geral computável dinâmico com gerações sobrepostas. Tal modelo se justifica porque permite
que se examinem, numa perspectiva intertemporal, os efeitos advindos de alterações tributárias sobre
o bem-estar das diferentes gerações de indivíduos que integram o modelo. Trata-se, portanto, de um
instrumental adequado para a análise de políticas tributárias porque, sabidamente, os seus efeitos se
estendem ao longo de vários períodos de tempo. Após esta Introdução, a Seção dois apresenta detalhadamente o modelo, bem como os procedi-
mentos usados para a obtenção dos seus principais parâmetros; a Seção três descreve as simulações
efetuadas e analisa resultados encontrados; e, finalmente, na Seção quatro aparecem as Considerações
Finais. 2.1. Famílias O modelo de Ando and Modigliani (1963) e Modigliani and Brumberg (1954) fornece a estrutura
teórica para a modelagem do comportamento das famílias quanto às suas decisões de consumo e lazer. De acordo com esse modelo, as famílias escolhem os níveis de consumo e lazer corrente e futuro em
função de suas expectativas de renda de todo o ciclo da vida. Quando jovens, considerando-se a falta
de experiência profissional e a baixa produtividade, se supõe que as famílias tenham uma renda relati-
vamente mais baixa. Na meia idade, quando atingem a maturidade profissional, elas conquistam o pico
de renda e, ao chegar à velhice, a sua renda tende a sofrer uma queda significativa. A trajetória da renda acima descrita configura um significativo grau de variação ao longo do ciclo
da vida. Os indivíduos, no entanto, tendem a manter uma alta estabilidade do consumo intertempo-
ralmente, o que significa que o comportamento da poupança depende do estágio do seu ciclo da vida. Assim, quando jovens, os indivíduos tendem a tomar empréstimos, na meia idade eles geram poupança
suficiente para pagar os empréstimos feitos nos períodos anteriores e financiar um padrão de consumo
para os períodos futuros. Na aposentadoria, sendo a renda do trabalho nula, as famílias consomem os
recursos que acumularam durante a vida. Com base no exposto, adota-se, no modelo, a hipótese de que
cada família maximiza a sua utilidade intertemporal com base na sua expectativa de renda ao longo do
ciclo da vida. U =
1
1 −1
γ
11
X
t=1
1
(1 + δ′)t−1 u(ct,lt)t−1
γ
u(ct,lt) =
c
1−1
ρ
t
+ αl
1−1
ρ
t
1
1−1/ρ
(1) (1) em que ct e lt representam o consumo e o lazer em cada período e os parâmetros γ,δ′,ρ e α represen-
tam, respectivamente, a elasticidade de substituição intertemporal do consumo nas diferentes idades,
a taxa de preferência pelo presente, a elasticidade de substituição entre o consumo e o lazer e a taxa de
preferência pelo lazer. tipo de doação entre gerações. A cada cinco anos, entendida como a unidade de tempo do modelo, a
geração mais velha desaparece e surge uma geração mais jovem e mais numerosa, o que configura a
existência de crescimento populacional. tipo de doação entre gerações. A cada cinco anos, entendida como a unidade de tempo do modelo, a
geração mais velha desaparece e surge uma geração mais jovem e mais numerosa, o que configura a
existência de crescimento populacional. A economia é aberta e composta por dois setores produtivos sendo que o primeiro produz um bem
demandado domesticamente, tanto para consumo quanto para investimento e o segundo produz um
bem destinado à exportação. As famílias, por sua vez, podem escolher entre o consumo de um bem
nacional ou um importado. Adota-se a suposição de pequeno país, significando que o preço das expor-
tações, em moeda externa, é exógeno e que a demanda externa por produtos nacionais é infinitamente
elástica. A balança comercial, no entanto, é endógena e sensível às condições internas de produção. Assim, de acordo com esta estrutura do modelo usado neste trabalho, as mudanças nas políticas fiscais
podem mudar o saldo comercial. Isto, por sua vez, influencia o montante de dívida externa, a poupança
macroeconômica e o estoque de capital. 2. ESTRUTURA DO MODELO Neste trabalho utiliza-se um modelo de equilíbrio geral computável dinâmico de gerações sobre-
postas.1 Em cada período, a economia inclui 11 classes etárias (gerações) de famílias, com cinco anos
cada, desconsiderando-se os períodos até os 21 e após os 75. As oito primeiras gerações são admitidas
ativas e as três últimas inativas (aposentadas) e não se considera a existência de herança ou qualquer 300 RBE
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com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas 2.1. Famílias A demanda agregada de consumo (ct) é composta pelo consumo do bem produzido nacionalmente
(Bem1) e pelo bem importado (Bem2): ct =
α1c
1−1
ρ1
1,t
+ (1 −α1)c
1−1
ρ1
2,t
1
1−1/ρ1
(2) (2) sendo ρ1 a elasticidade de substituição do consumo entre o Bem1 e o Bem2 e α1 o parâmetro que
indica a preferência pelo bem produzido nacionalmente em relação ao bem importado. sendo ρ1 a elasticidade de substituição do consumo entre o Bem1 e o Bem2 e α1 o parâmetro que
indica a preferência pelo bem produzido nacionalmente em relação ao bem importado. 301 RBE
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Jul-Set 2009 Carlos Renato Salami e Adelar Fochezatto Em cada período, o tempo total à disposição das famílias para alocação entre consumo e lazer é
normalizado igual a um, sendo as famílias pertencentes às três últimas classes de idade consideradas
inativas. Tem-se, portanto, que 0 < lt ≤1∀t = 1, . . . ,8 e l9,l10 e l11 = 1, com (1 −lt) representando
o tempo destinado ao trabalho. A restrição orçamentária intertemporal a que cada família está sujeita
é dada por: w(1 −τw)(1 −τr)
8
X
t=1
ht(1 −lt)
(1 + rm)t−1 + (1 −τr)
11
X
t=9
PRt
(1 + rm)t−1 = p
11
X
t=1
ct
(1 + rm)t−1
(3) (3) sendo τw a contribuição social salarial, τr o imposto de renda e PRt as transferências a título de
aposentadoria deduzidas do imposto de renda. O parâmetro ht indica a evolução do salário médio (w) recebido por cada família ao longo de toda a
sua vida ativa. Considera-se que, na juventude, as famílias recebam um salário relativamente inferior
ao que receberão ao decorrer dos anos, com o aumento da sua qualificação e produtividade. Conforme
proposto por Auerbach and Kotlikoff (1987), é utilizado neste trabalho o seguinte perfil de remuneração,
expresso em termos dos anos de experiência: ht = exp(a+bt+ct2), com a restrição de que P8
t=1 ht =
1. O fator de desconto 1/(1 + rm) considera a taxa de juros relevante para as famílias (rm), a qual
deduz dos juros de mercado o imposto sobre a renda do capital (τe). Assim, rm = r(1 −τe). 2.1. Famílias O preço p
representa o índice de preços ao consumidor, sendo expresso pela seguinte equação: p = (1 + τ)
αρ1
1 p1−ρ1
1
+ (1 −α1)ρ1p1−ρ1
2
1
1−ρ1
(4) (4) na qual τ representa o imposto sobre o consumo de bens nacionais ou importados. Assim, a restrição
orçamentária apresenta a seguinte propriedade: pct = (1 + τ) (p1c1,t + p2c2,t)
(5) (5) Formalmente, considera-se que as famílias maximizam a expressão (1) sujeitas à expressão (3). As
condições de primeira ordem são as seguintes: Formalmente, considera-se que as famílias maximizam a expressão (1) sujeitas à expressão (3). As
condições de primeira ordem são as seguintes: u(ct,lt)
1
ρ −1
γ c
−1
ρ
t
= λp
1 + δ′
1 + rm
t−1
(6)
u(ct,lt)
1
ρ −1
γ αl
−1
ρ
t
= λw(1 −τw)(1 −τr)ht
1 + δ′
1 + rm
t−1
+ µt
(7) u(ct,lt)
1
ρ −1
γ c
−1
ρ
t
= λp
1 + δ′
1 + rm
t−1
(6) (6) u(ct,lt)
1
ρ −1
γ αl
−1
ρ
t
= λw(1 −τw)(1 −τr)ht
1 + δ′
1 + rm
t−1
+ µt
(7) (7) sendo λ o multiplicador associado à restrição orçamentária e µt, com t = 1, . . . ,8, os multiplicadores
associados às restrições impostas sobre o lazer. Conforme Schubert and Letournel (1991), a presença da
limitação imposta sobre o lazer, a saber, de que l9,l10 e l11 = 1, impede a resolução analítica do sistema. Entretanto, se são considerados apenas os períodos ativos, as expressões precedentes conduzem às
seguintes relações: lt = ct
αp
w(1 −τw)(1 −τr)ht
ρ
(8)
lt = l1
1 + αρ(p/(w(1 −τw)(1 −τr)ht))ρ−1
1 + αρ(p/(w(1 −τw)(1 −τr)ht))ρ−1
γ−ρ
ρ−1
. 1 + rm
1 + δ′
γ(t−1)
(9)
ct = c1
1 + αρ(p/(w(1 −τw)(1 −τr)ht))ρ−1
1 + αρ(p/(w(1 −τw)(1 −τr)ht))ρ−1
γ−ρ
ρ−1
. 1 + rm
1 + δ′
γ(t−1)
(10) lt = ct
αp
w(1 −τw)(1 −τr)ht
ρ
(8) (8) lt = l1
1 + αρ(p/(w(1 −τw)(1 −τr)ht))ρ−1
1 + αρ(p/(w(1 −τw)(1 −τr)ht))ρ−1
γ−ρ
ρ−1
. 1 + rm
1 + δ′
γ(t−1)
(9)
ct = c1
1 + αρ(p/(w(1 −τw)(1 −τr)ht))ρ−1
1 + αρ(p/(w(1 −τw)(1 −τr)ht))ρ−1
γ−ρ
ρ−1
. 1 + rm
1 + δ′
γ(t−1)
(10) (9) (10) 302 RBE
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Jul-Set 2009 2.1. Famílias A partir dos dados fiscais do Brasil, pode-se dizer que o montante
transferido às famílias corresponde, em média, a 70% da renda recebida ao longo da sua vida ativa, o
que é expresso da seguinte forma: PR = 0,7w
8
8
X
t=1
ht(1 −lt)
(16) (16) As contribuições sociais dos trabalhadores e dos empregadores para a previdência são dadas por: As contribuições sociais dos trabalhadores e dos empregadores para a previdência são dadas por: CSR = PR
11
X
t=9
(1 + n)1−t =
τwr + τ
′′
r
w
8
X
t=1
ht(1 −lt)(1 + n)1−t
(17) (17) onde, τwr e τ
′′
r representam as contribuições sociais a título de aposentadoria dos trabalhadores e dos
empregadores, respectivamente. onde, τwr e τ
′′
r representam as contribuições sociais a título de aposentadoria dos trabalhadores e dos
empregadores, respectivamente. 2.1. Famílias 3 / p. 299–314
Jul-Set 2009 RBE
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Jul-Set 2009 Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
B
M d l d E
ilíb i G
l d G
õ
S b
t com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas Conhecida a demanda de consumo agregado (ct), as demandas dos dois bens podem ser expressas
como segue: c1,t = ct
α1p
(1 + τ)p1
ρ1
(11)
c2,t = ct
α1p
(1 + τ)p2
ρ1
(12) (11) (12) Em um modelo de gerações sobrepostas, uma condição necessária para que a poupança agregada
seja positiva é que haja crescimento populacional. Isto porque, neste caso, a magnitude da poupança
gerada pelos jovens supera a poupança negativa dos inativos. Admitindo-se P como sendo a população
total da economia em um dado momento e n a sua taxa de crescimento, a população da classe etária t,
para t = 1, . . . ,11, é dada por: n(1 + n)11−t
(1 + n)11 −1P
(13) (13) O modelo é escrito em termos do número de indivíduos da geração mais nova, que compreende
n(1+n)10
(1+n)11−1P pessoas. Assim, o consumo total pode ser expresso como sendo: C = (1 + n)11 −1
n(1 + n)10
1
P
X n(1 + n)11−t
(1 + n)11 −1Pct
(14)
C =
11
X
t=1
ct
(1 + n)t−1
(15) (14) (15) Considera-se, ainda, que, ao longo da sua vida ativa, as famílias contribuem para fins de aposen-
tadoria, na forma de pagamentos para a previdência social, e que recebem transferências a título de
aposentadoria quando inativos. 2.2. Empresas A partir da resolução do problema de
maximização do lucro das empresas, obtêm-se as seguintes expressões para as demandas por capital e
trabalho e para a fronteira dos preços dos fatores.2 Ki
=
YiAσi−1
i
θipi
r + δ
σi
(20)
Li
=
YiAσi−1
i
(1 −θi)pi
s(1 −τ
′′
r )
σi
(21)
(piAi)1−σi
=
θσi
i (r + δ)1−σi + (1 −θ)σi(s(1 + τ
′′
r ))1−σi
(22) (20) (21) (piAi)1−σi
=
θσi
i (r + δ)1−σi + (1 −θ)σi(s(1 + τ
′′
r ))1−σi
(22) (22) No que tange ao mercado de trabalho, tem-se que a relação entre o salário médio pago pelos em-
pregadores (s) e o salário médio recebido pelos trabalhadores ao longo da sua vida ativa é definida da
seguinte forma: s
8
X
t=1
1 −lt
(1 + n)t−1 = w
8
X
t=1
ht(1 −lt)
(1 + n)t−1
(23) (23) 2.3. Governo 2Conforme Schubert and Letournel (1991), a fronteira dos preços dos fatores ou condição de lucro nulo decorre da hipótese
da constância dos rendimentos de escala, o que determina que a relação entre os preços dos fatores depende apenas dos
parâmetros da função de produção. 2.2. Empresas A economia é composta de dois setores produtivos, sendo que o setor 1 produz para o mercado
doméstico e o setor 2 para o mercado externo. Supõe-se, ademais, que a produção dos dois setores seja
determinada por uma função de produção CES, com rendimentos de escala constantes. 303 RBE
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Jul-Set 2009 Carlos Renato Salami e Adelar Fochezatto Yi = Fi(Ki,Li) = Ai
θiK
1−1
σi
i
+ (1 −θi)L
1−1
σi
i
1
1−1
σi
para
i = 1,3
(18) (18) As variáveis Yi,Ki e Li representam, respectivamente, a produção, o capital e o nível de emprego do
setor i. O parâmetro Ai é considerado um fator de escala e representa a produtividade total dos fatores. O parâmetro σi expressa a elasticidade de substituição entre capital e trabalho e θi a participação do
capital na função de produção. As empresas maximizam seus lucros intertemporalmente, sujeitas à restrição advinda dos custos de
produção: Πi =
piYi −s(1 + τ
′′
r )Li −pi(r + δ)Ki
(1 −τ ′)
para
i = 1,3
(19) (19) onde, p1 e p3 correspondem aos preços dos bens 1 e 3, r é a taxa de juros real, δ a taxa de depreciação
do capital, s o salário médio pago pelas empresas, τ
′′
r a contribuição social paga pelos empregadores e
τ ′ o imposto sobre o lucro. O Bem1 é utilizado também como bem de capital sendo a importação desse tipo de bem nula. Embora as empresas estejam sujeitas aos custos de ajustamento do capital no estado transitório, estes
desaparecem no estado estacionário de longo prazo. Nestas circunstâncias, o investimento compreende,
de um lado, a depreciação do capital e, de outro, o crescimento do estoque de capital à taxa de cresci-
mento populacional. Tem-se, portanto que Ii = (δ + n)Ki. 2.3. Governo O Estado arrecada receitas fiscais e é consumidor tanto de bens produzidos nacionalmente quanto
de bens importados: RF −(p1G1 + p2G2) = (r −n)D
(24) (24) sendo RF o montante de receitas fiscais, G1 e G2 as despesas com os bens 1 e 2 e D o montante da
dívida pública, na forma de títulos públicos. 2Conforme Schubert and Letournel (1991), a fronteira dos preços dos fatores ou condição de lucro nulo decorre da hipótese
da constância dos rendimentos de escala, o que determina que a relação entre os preços dos fatores depende apenas dos
parâmetros da função de produção. 304 RBE
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com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
Bas
M d l d Eq ilíbri G ral d G ra õ s S br
stas Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas A capacidade de financiamento do Estado lhe permite o pagamento dos encargos sobre a dívida,
representados pela diferença entre a taxa de juros real e a taxa de crescimento populacional. A função
receita fiscal explicitando as diferentes fontes de receitas do Estado é definida pela seguinte equação: RF = τrw(1−τw)
8
X
t=1
ht(1 −lt)
(1 + n)t−1 +τrPR
11
X
i=9
1
(1 + n)t−1 +τ(p1CB1 +p2CB2)+rτe
E
1 + n (25) sendo E definido como a soma ponderada dos montantes de poupança de um consumidor pertencente
a cada classe etária, Et. Tem-se, portanto, que: sendo E definido como a soma ponderada dos montantes de poupança de um consumidor pertencente
a cada classe etária, Et. 2.3. Governo Tem-se, portanto, que: E =
10
X
i=1
Et
(1 + n)t−1
(26
E1 = w(1 −τw)(1 −τr)h1(1 −l1) −pc1
Et = w(1 −τw)(1 −τr)ht(1 −lt) + (1 + rm)Et−1 −pct; t = 2, · · · ,8
Et = PR(1 −τr) + (1 + rm)Et−1 −pct; t = 9,10 Et
(1 + n)t−1
(26) (26) E1 = w(1 −τw)(1 −τr)h1(1 −l1) −pc1
Et = w(1 −τw)(1 −τr)ht(1 −lt) + (1 + rm)Et−1 −pct; t = 2, · · · ,8
Et = PR(1 −τr) + (1 + rm)Et−1 −pct; t = 9,10 A poupança líquida da economia é calculada, considerando-se a poupança negativa das gerações
mais velhas, da seguinte forma: EM =
1 −
1
1 + n
E
(27) (27) 4De acordo com a Lei de Walras, a soma dos valores dos excessos de demanda agregados na economia deve ser igual à zero.
Assim, em uma economia com n mercados, se n −1 estiverem em equilíbrio, o n-ésimo também estará. Todas as igualdades em consideração se referem ao estado estacionário. 3Todas as igualdades em consideração se referem ao estado estacionário. 2.4. Equilíbrio dos mercados Formalizado o comportamento de cada agente econômico que integra o modelo, passa-se, a seguir,
a considerar as condições de equilíbrio que devem ser satisfeitas, em cada um dos mercados, para que
seja obtida a resolução numérica do modelo.3 Serão explicitadas as condições de equilíbrio do mercado
de trabalho, do mercado do Bem1 e da balança comercial, sendo o equilíbrio do mercado de capitais
definido pela lei de Walras.4 No mercado de trabalho, o equilíbrio é expresso pela igualdade entre a oferta e a demanda de tra-
balho. L1 + L3 =
8
X
t=1
1 −lt
(1 + n)t−1
(28) (28) onde, L1 e L3 representam as demandas de trabalho nos setores produtores dos bens 1 e 3, respectiva-
mente, e a expressão do lado direito representa a oferta de trabalho total das oito gerações ativas que
compõem o modelo. No mercado do Bem1, produzido nacionalmente, supõe-se que seja satisfeita a seguinte condição
de equilíbrio: Y1 = CB1 + (n + δ)(K1 + K3) + G1
(29) (29) sendo n + δ)(K1 + K3) a demanda de investimento dos dois setores produtivos e CB1 o consumo
total do Bem1. sendo n + δ)(K1 + K3) a demanda de investimento dos dois setores produtivos e CB1 o consumo
total do Bem1. 3Todas as igualdades em consideração se referem ao estado estacionário. 4De acordo com a Lei de Walras, a soma dos valores dos excessos de demanda agregados na economia deve ser igual à zero. Assim, em uma economia com n mercados, se n −1 estiverem em equilíbrio, o n-ésimo também estará. 305 RBE
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Jul-Set 2009 RBE
Rio de Janeiro
v. 63 n. 3 / p. 299–314
Jul-Set 2009 Carlos Renato Salami e Adelar Fochezatto O saldo efetivo da balança comercial é o seguinte: O saldo efetivo da balança comercial é o seguinte: O saldo efetivo da balança comercial é o seguinte: BC = p3Y3 −p2(CB2 + G2)
(30) (30) No que se refere ao relacionamento da economia com o resto do mundo, considera-se que o Brasil
é uma pequena economia aberta. Assim, o preço é dado pelo mercado internacional e a demanda por
exportações é infinitamente elástica. Neste caso, sob a hipótese de perfeita mobilidade de capitais, o
país não exerce nenhuma influência sobre a taxa de juros mundial, sendo, portanto, a taxa de juros
doméstica exógena e igual à taxa de juros mundial. Sendo DX a dívida externa do país, no estado estacionário satisfaz-se a seguinte relação: (r −n)DX = BC
(31) (31) Finalmente, pela lei de Walras, obtém-se o equilíbrio do mercado de capitais: Finalmente, pela lei de Walras, obtém-se o equilíbrio do mercado de capitais: E
(1 + n) = EM
n
= (K1 + K3) + (D −DX)
(32) (32) Tem-se, portanto, que a poupança líquida financia o estoque de capital da economia, bem como o
excedente da dívida pública em relação à dívida externa. Tem-se, portanto, que a poupança líquida financia o estoque de capital da economia, bem como o
excedente da dívida pública em relação à dívida externa. O conjunto de equações apresentado nesta seção possibilita que se tenha uma idéia do comporta-
mento dos diferentes agentes e do funcionamento dos diferentes mercados que integram o modelo. A seguir, proceder-se-á à parametrização e à calibragem dos parâmetros necessários para a resolução
numérica do modelo. 5Excluindo a base monetária. 2.5. Calibragem do modelo Nos modelos de equilíbrio geral computável, a calibragem consiste no procedimento que possibilita
a reprodução dos dados do ano base como solução do modelo. Isto exige a construção de um amplo e
consistente banco de dados. Ele inclui os valores de todas as variáveis do modelo, geralmente expressos
na forma de uma matriz de contabilidade social, elaborada a partir das contas nacionais e de diversas
outras fontes estatísticas. A calibragem consiste na resolução das equações na forma inversa, sendo os
valores dos parâmetros calculados por meio da fixação dos valores das variáveis endógenas nos níveis
observados no período de referência. Além disso, é necessário especificar valores de alguns parâmetros
não passíveis de calibragem, os quais são tirados da literatura pertinente. Os dados utilizados na calibragem do modelo correspondem à média dos cinco anos compreendidos
entre 1994 e 1998, período de estabilidade cambial. Os principais agregados macroeconômicos, extraí-
dos das contas nacionais e de outras fontes, utilizados no modelo estão na Tabela 1. De acordo com
os dados do IBGE, a despesa do setor público consolidado, que inclui os gastos da administração direta
federal, dos estados, dos municípios, das empresas públicas, do banco central e da seguridade social,
atingiu, em média, 27,6% do PIB no período, dos quais o pagamento de benefícios pelo Regime Geral
da Previdência Social representou, aproximadamente, 4,6% do PIB. Dessa forma, pela ótica do resultado
primário, o setor público apresentou uma situação de equilíbrio fiscal. Entretanto, após a inclusão dos
gastos com o pagamento de juros sobre a dívida pública, de aproximadamente 4,7% do PIB, o equilíbrio
primário transforma-se em um déficit da ordem de 4,5% do PIB. Outras variáveis importantes são: a
dívida líquida do setor público e a taxa real de juros. O primeiro representou, em média, 25,6% do PIB
no período.5 Em relação aos juros, foi utilizada a média da taxa de longo prazo, que ficou em 16,5% no
período. Em relação aos setores produtivos que integram o modelo, supõe-se que os dois setores utilizam
igual tecnologia. Isto significa que eles possuem as mesmas elasticidades de substituição (σ1 = σ3 = 306 RBE
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Jul-Set 2009 Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas Table 1: Principais agregados macroeconômicos do modelo (% do PIB)
Agregados macroeconômicos
Participação no PIB (%)
Fonte
Informações provenientes das contas nacionais
Consumo final das famílias (C)
61,6
IBGE
Consumo final da administração pública (G)
18,7
IBGE
Formação bruta de capital (I)
21,5
IBGE
Exportações (X)
7,6
IBGE
Importações (M)
9,4
IBGE
Informações provenientes de outras fontes estatísticas
Estoque de capital
300
Lledo (2001)
Carga tributária (exclusive seguridade social)
21,43
SRF
Contribuições para a seguridade social
6,38
MPAS
Gastos do governo com benefícios
4,6
MPAS
Pagamento de juros sobre a dívida pública
4,7
SRF
Dívida pública total do setor público
34,1
SRF
Fonte: IBGE, MPAS, SRF, Lledo (2001). Table 1: Principais agregados macroeconômicos do modelo (% do PIB) σ), a mesma participação do capital na função de produção (θ1 = θ3 = θ) e as mesmas produtividades
totais dos fatores (A1 = A3 = A). Tais hipóteses implicam na igualdade dos preços praticados pelos
dois setores produtivos: p1 = p3. σ), a mesma participação do capital na função de produção (θ1 = θ3 = θ) e as mesmas produtividades
totais dos fatores (A1 = A3 = A). Tais hipóteses implicam na igualdade dos preços praticados pelos
dois setores produtivos: p1 = p3. O preço do Bem1 é utilizado como numerário, sendo, portanto, p1 = 1 e o do Bem2 ( ˙p2) é fixado
pelo resto do mundo, em moeda estrangeira. Assim, sendo e a taxa de câmbio, o preço do Bem2, em
moeda doméstica, é p2 = e ˙p2. Além disso, impondo-se que ˙p2 = 1, resulta que p2 = e. Finalmente, o
preço do Bem3 se expressa como p3 = e ˙p3, sendo ˙p3 o preço das exportações em moeda estrangeira. Portanto, da mesma forma, considerando-se que ˙p3 = 1, tem-se que p3 = e. O perfil de remuneração individual descreve os salários relativos por idade. Auerbach and Kotlikoff
(1987) definem em seu trabalho o perfil de remuneração do trabalho como uma função exponencial dos
anos de experiência, da seguinte forma: et = exp(a + bt + ct2). A partir desta especificação funcional,
se utiliza as estimativas obtidas por Ferreira and Araújo (1999),6 o qual obteve os seguintes valores para
os parâmetros: a = −0,231, b = 0,05 e c = −0,0009. 6Ferreira and Araújo (1999) estimam os valores dos parâmetros que refletem o retorno da experiência de residentes em área
urbana com idade entre 25 e 65 anos, utilizando a Pesquisa Nacional por Amostra de Domicílios (PNAD). 2.5. Calibragem do modelo A partir destas estimativas foi feita a calibragem
dos parâmetros pertencentes ao perfil de remuneração, os quais aparecem na Tabela 2. Table 2: Parâmetros que definem o perfil de remuneração do trabalho
h1
h2
h3
h4
h5
h6
h7
h8
0,79
0,85
0,90
1,0
1,05
1,09
1,14
1,18
Fonte: Cálculo dos autores. Table 2: Parâmetros que definem o perfil de remuneração do trabalho Apesar de ht e w serem exógenos, pela expressão 23 o salário efetivo torna-se endógeno e depen-
dente do lazer, lt, que, por sua vez, depende do preço dos produtos e dos impostos. 6Ferreira and Araújo (1999) estimam os valores dos parâmetros que refletem o retorno da experiência de residentes em área
urbana com idade entre 25 e 65 anos, utilizando a Pesquisa Nacional por Amostra de Domicílios (PNAD). 307 RBE
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v. 63 n. 3 / p. 299–314
Jul-Set 2009 Carlos Renato Salami e Adelar Fochezatto Os parâmetros calculados pelo processo de calibragem e os obtidos diretamente de outras fontes
estão na Tabela 3. O modelo inclui, ainda, os parâmetros fiscais e previdenciários que compõem a matriz tributária
do Brasil. Para ajustar ao modelo usado, procedeu-se a agregação dos diferentes impostos em quatro
categorias. A Tabela 4 apresenta as alíquotas destas categorias. Table 3: Valores dos parâmetros usados no modelo7
Parâmetros
Valores
Fonte
Preferência das famílias pelos bens na-
cionais (α1)
0,77
Calibragem
Preferência das famílias pelo lazer (α)
0,72
Calibragem
Participação do capital na produção (θ)
0,97
Calibragem
Produtividade total dos fatores (A)
3,71
Calibragem
Contribuição previdenciária dos empre-
gadores (τ
′′
r )
0,11
Calibragem
Elasticidade de substituição capital tra-
balho (σ)
0,47
Mercenier and Sampaio de Souza (1994)
Taxa de depreciação do capital (δ)
5,0 % a.a. Oliveira and Lannes Jr (2000)
Taxa de preferência pelo presente (δ′)
2,1 % a.a. Lledo (2001)
Elasticidade de substituição intertemporal
do consumo (γ)
0,40
Lledo (2001)
Elasticidade de substituição entre consumo
e lazer (ρ)
1,15
Lledo (2001)
Elasticidade de substituição entre o Bem1
e o Bem2 (ρ1)
1,41
Tourinho et al. (2002)
Taxa de crescimento populacional (n)
1,90 % a.a. IBGE (1994,1998)
Taxa de juro real (r)
7,54 % a.a. Ipeadata
Fonte: Elaboração dos autores. 3. SIMULAÇÕES E ANÁLISE DOS RESULTADOS Determinado o equilíbrio estacionário de referência, serão procedidas a seguir algumas simulações
envolvendo alterações na política fiscal do governo.9 É importante notar que, com exceção das duas
primeiras simulações, as demais são efetuadas a partir de modificações fiscais que contemplam a
manutenção das receitas fiscais no nível anterior à mudança. Tal imposição facilita a análise com-
parativa das diferentes simulações. Esta condição determina que a dívida interna permaneça estável. Isto porque, no caso da economia aberta, não há a possibilidade das modificações fiscais provocarem
variações na taxa de juros doméstica, a qual tende a igualar-se à taxa mundial. É oportuno salientar também que não é intenção, com as simulações, tentar determinar o sistema
fiscal que fornece o montante de receitas fiscais ótimo, no sentido de induzir o maior nível de bem-estar
possível. O que se pretende é efetuar algumas alterações marginais na estrutura tributária atual da
economia com o intuito de se analisar as suas influências sobre a atividade econômica no longo prazo. As simulações são as seguintes: Sim-1) redução de 9,81% na alíquota do imposto sobre o valor agregado; Sim-1) redução de 9,81% na alíquota do imposto sobre o valor agregado; Sim-2) redução de 14,77% no imposto de renda; Sim-3) redução de 9,81% na alíquota do imposto sobre o valor agregado, compensada pela introdução
de um imposto lump-sum; Sim-3) redução de 9,81% na alíquota do imposto sobre o valor agregado, compensada pela introdução
de um imposto lump-sum; Sim-4) redução de 14,77% no imposto de renda, compensada pela introdução de um imposto lump-sum;
e Sim-4) redução de 14,77% no imposto de renda, compensada pela introdução de um imposto lump-sum;
e Sim-5) redução de 9,81% do imposto sobre o valor agregado, compensada pelo aumento de 14,78% no
imposto de renda. Sim-5) redução de 9,81% do imposto sobre o valor agregado, compensada pelo aumento de 14,78% no
imposto de renda. As duas primeiras simulações resultam em um nível inferior de arrecadação tributária de exata-
mente 5%. Objetiva-se, com estas simulações, verificar a proposição comumente defendida de que a
redução de impostos geraria um estímulo à atividade econômica cujo impacto na arrecadação seria su-
ficiente para compensar a redução inicial do imposto. Nas três últimas simulações, considera-se que
a redução de um imposto seja compensada, sob o ponto de vista das receitas, pela elevação de outro
imposto. 3. SIMULAÇÕES E ANÁLISE DOS RESULTADOS Pretende-se, com estas três últimas simulações, fazer uma análise comparativa das diferentes
opções fiscais em termos de impactos favoráveis sobre a economia. Os resultados de todas as simu-
lações são apresentados na Tabela 5 e, como ela é auto-explicativa, analisam-se apenas os resultados
considerados mais relevantes. 2.5. Calibragem do modelo Table 3: Valores dos parâmetros usados no modelo7 Table 4: Alíquotas tributárias efetivas ajustadas à estrutura do modelo8 Table 4: Alíquotas tributárias efetivas ajustadas à estrutura do modelo8
Impostos
Alíquotas (%)
Imposto sobre o consumo
τ = 15,68
Imposto sobre o rendimento da poupança
τe = 13,21
Imposto sobre a renda
τr = 11,82
Contribuição previdenciária salarial
τwr = 4,03
Fonte: Cálculo dos autores. 7Os parâmetros θ,A e α1 são obtidos a partir das seguintes expressões: A = [θσ(r + δ)1−σ + (1 −θ)σ(s(1 +
τ
′′))1−σ]1/1−σ. p1; θ =
»“
K
L
”1/σ
. r+δ
s(1+τ′′
r )
– . »
1 +
“
K
L
”1/σ
. r+δ
s(1+τ′′
r )
–
; e α1 = 1
. »
1 +
“
c2
c1
”1/ρ1–
. 7Os parâmetros θ,A e α1 são obtidos a partir das seguintes expressões: A = [θσ(r + δ)1−σ + (1 −θ)σ(s(1 +
τ
′′))1−σ]1/1−σ. p1; θ =
»“
K
L
”1/σ
. r+δ
s(1+τ′′
r )
– . »
1 +
“
K
L
”1/σ
. r+δ
s(1+τ′′
r )
–
; e α1 = 1
. »
1 +
“
c2
c1
”1/ρ1–
. 308 RBE
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Jul-Set 2009 RBE
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v. 63 n. 3 / p. 299–314
Jul-Set 2009 Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas 8No cálculo das alíquotas efetivas são consideradas as seguintes bases de incidência tributária expressas em percentual do
PIB, obtidas a partir das contas nacionais: consumo das famílias (61,6), excedente operacional bruto (41,2), salários (38,5),
remuneração dos autônomos (5,7), poupança (18,1) e depreciação (3% a.a.).
9Para isso utiliza-se o software SORITEC, o qual emprega o algoritmo de resolução Newton. 9Para isso utiliza-se o software SORITEC, o qual emprega o algoritmo de resolução Newton. Carlos Renato Salami e Adelar Fochezatto Em relação aos inativos, mesmo inexistindo o efeito-substituição, verifica-se que o consumo também
se expande. Neste caso, a magnitude do resultado também é influenciada pelo fato de se beneficiarem
relativamente mais do que os ativos da redução do imposto sobre o consumo. No agregado, o consumo
cresce em volume, sendo que o bem produzido para o mercado interno e o importado apresentam a
mesma taxa de crescimento. Em relação à produção, verifica-se que a expansão do consumo desloca o esforço produtivo para
o atendimento do mercado interno em detrimento do externo. Dada a maior participação relativa do
setor que produz para o mercado interno, a expansão da produção deste setor mais do que compensa
a queda do setor exportador, fazendo com que o PIB cresça. Acompanhando o crescimento do PIB, o
emprego também cresce. A redução das exportações e o aumento das importações, gerados pela expansão do consumo,
causam, por seu turno, a ampliação do déficit comercial e o aumento do endividamento externo. A
expansão da poupança externa, por sua vez, possibilita financiar a expansão do investimento, mesmo
com a diminuição da poupança nacional. A redução das receitas fiscais em 5%, mantendo os gastos
do governo, acaba provocando o aumento da relação dívida/PIB. Vê-se, portanto, que do ponto de vista
fiscal, o efeito expansivo gerado pela redução do imposto não foi suficiente para compensar a queda na
arrecadação tributária. A redução das exportações e o aumento das importações, gerados pela expansão do consumo,
causam, por seu turno, a ampliação do déficit comercial e o aumento do endividamento externo. A
expansão da poupança externa, por sua vez, possibilita financiar a expansão do investimento, mesmo
com a diminuição da poupança nacional. A redução das receitas fiscais em 5%, mantendo os gastos
do governo, acaba provocando o aumento da relação dívida/PIB. Vê-se, portanto, que do ponto de vista
fiscal, o efeito expansivo gerado pela redução do imposto não foi suficiente para compensar a queda na
arrecadação tributária. 3.2. Simulação 2: Redução do imposto sobre a renda Diferentemente do ocorrido na situação anterior, neste caso não se verifica o efeito inicial sobre os
preços. Entretanto, a redução da alíquota do imposto de renda acarreta uma maior expansão da oferta
de trabalho do que no caso anterior. Como conseqüência, o consumo da população ativa se expande a
uma taxa superior à verificada anteriormente. Deve-se considerar que, na primeira simulação, o efeito-
substituição gerado pela redução do imposto ocorre por meio da queda dos preços e da conseqüente
elevação do salário real, sendo limitado, portanto, pela magnitude da propensão a consumir. No caso
em consideração, a redução do imposto exerce um efeito direto sobre a renda, tornando mais evidente
para as famílias a mudança no preço relativo do trabalho e do lazer. No que se refere ao consumo, portanto, verifica-se, como na simulação anterior, que tanto o dos
ativos quanto dos inativos se expande. Entretanto, conforme já mencionado, a expansão do consumo
dos ativos é explicada pela ocorrência dos efeitos renda e substituição, enquanto que a dos inativos
ocorre somente em função do segundo efeito. No que concerne à produção, tendo em vista a maior expansão do consumo interno, observa-se um
aprofundamento da transferência do esforço produtivo do setor exportador para o setor que produz
para o mercado doméstico. Tal fato propicia um maior crescimento do PIB e do emprego do que no caso
anterior. O saldo da balança comercial e o endividamento externo também apresentam desempenhos
relativamente piores. 3.1. Simulação 1: Redução do imposto sobre o valor agregado A redução do imposto sobre o valor agregado apresenta como impacto inicial a diminuição do preço
do consumo interno (p), o que favorece a expansão do consumo da população ativa em virtude de dois
efeitos. Em primeiro lugar, porque a redução do preço causa a elevação do salário real (w/p), possi-
bilitando aos assalariados desfrutar de um nível mais elevado de consumo. Por outro lado, à medida
que o lazer se torna relativamente mais caro, os trabalhadores são incentivados a substituir lazer por
trabalho, expandindo a oferta de trabalho, a qual cresce em torno de 4% em relação ao equilíbrio de
referência. 309 RBE
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Jul-Set 2009 RBE Carlos Renato Salami e Adelar Fochezatto : Fonte: Cálculos dos autores. (∗)Os valores expressam a diferença percentual em relação ao equilíbrio
de referência; (∗∗)em relação ao PIB. 3.3. Simulação 3: Redução do imposto sobre o valor agregado compensada pela
introdução de um imposto lump-sum Conforme já comentado, a redução do imposto sobre o valor agregado tem como efeito inicial a
redução do preço do consumo interno, o que aumenta o poder de compra das famílias. Entretanto, 310 RBE
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v. 63 n. 3 / p. 299–314
Jul-Set 2009 Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas Table 5: Resultados das simulações envolvendo alterações de política fiscal (∗)
Variável
Sim-1
Sim-2
Sim-3
Sim-4
Sim-5
Famílias
Bem-estar
2,62
3,67
-0,55
0,76
-1,52
Consumo do Bem1
0,46
0,68
0,02
0,24
-0,23
Consumo do Bem2
0,46
0,68
0,02
0,24
-0,23
Consumo dos ativos
1,59
2,30
0,07
0,83
-0,83
Consumo dos inativos
0,62
0,90
0,001
0,30
-0,33
Consumo agregado
0,46
0,68
0,02
0,27
-0,23
Lazer
-1,46
2,13
-1,54
-2,19
0,73
Poupança nacional
-3,66
3,46
-1,56
-1,34
-0,19
Empresas
Produção do Bem1
0,34
0,50
0,17
0,32
-0,17
Produção do Bem3
-0,32
0,47
6,21
5,94
0,11
Produção total
0,28
0,41
0,72
0,84
-0,14
Emprego
0,25
0,37
0,69
0,80
-0,13
Investimento
0,28
0,41
0,72
0,84
-0,14
Balança comercial (∗∗)
-0,07
-0,11
0,57
0,52
0,03
Preços
Preço do consumo
-1,33
0,00
-1,33
0,00
-1,33
Salário médio famílias
-0,06
-0,09
0,01
-0,02
0,03
Salário médio empresas
0,05
0,07
0,05
0,07
-0,02
Taxa de juros
0,00
0,00
0,00
0,00
0,00
Taxa de juros das famílias
0,00
0,00
0,00
0,00
0,00
Impostos
Valor agregado
14,1382
15,6754
14,1382
15,6754
14,1382
Renda
11,8161
10,0713
11,8161
10,0713
13,5631
Lump-sum
não
não
sim
sim
não
Poupança
13,2146
13,2146
13,2146
13,2146
13,2146
Equilíbrio do mercado de capitais (∗)
△EM/n
Y
-3,01
-2,86
-1,29
-1,11
-0,15
△(K1+K3)
Y
0,17
0,25
0,43
0,50
-0,09
△D
Y
3,34
3,34
0,00
0,00
0,00
△DX
Y
0,22
0,32
-1,67
-1,54
-0,10
: Fonte: Cálculos dos autores. (∗)Os valores expressam a diferença percentual em relação ao equilíbrio Table 5: Resultados das simulações envolvendo alterações de política fiscal (∗) 311 RBE
Rio de Janeiro
v. 63 n. 3 / p. 299–314
Jul-Set 2009 Carlos Renato Salami e Adelar Fochezatto tem-se, agora, com a introdução do imposto lump-sum, a redução da renda familiar, o que acaba por
limitar a expansão deste consumo. O resultado líquido é uma expansão do mesmo, embora a uma taxa
inexpressiva. Verifica-se, ademais, que os inativos aumentam menos o consumo do que os ativos, o
que se explica pelo fato de que a carga do imposto lump-sum é relativamente mais pesada para eles. A
evolução do consumo de produtos nacionais e importados é similar, já que a taxa de câmbio não varia,
não havendo, portanto, efeito substituição ocasionado pelos preços. tem-se, agora, com a introdução do imposto lump-sum, a redução da renda familiar, o que acaba por
limitar a expansão deste consumo. O resultado líquido é uma expansão do mesmo, embora a uma taxa
inexpressiva. Verifica-se, ademais, que os inativos aumentam menos o consumo do que os ativos, o
que se explica pelo fato de que a carga do imposto lump-sum é relativamente mais pesada para eles. A
evolução do consumo de produtos nacionais e importados é similar, já que a taxa de câmbio não varia,
não havendo, portanto, efeito substituição ocasionado pelos preços. As empresas, por seu turno, são estimuladas a contratar e a investir mais com vistas à satisfação da
demanda adicional. Todavia, diferentemente do verificado nas simulações precedentes, na situação em
questão, o esforço produtivo se direciona principalmente para o setor produtor de bens exportáveis. Isto
ocorre porque, enquanto as empresas do setor que produz para o mercado doméstico são restringidas
pela demanda das famílias, as empresas do setor exportador podem aumentar a sua produção, já que
sua demanda é perfeitamente elástica. Como resultado, o saldo comercial melhora e a dívida externa é
reduzida. Em se tratando da dívida interna, observa-se que ela permanece estável em virtude da hipótese
adotada de manutenção da receita tributária estável. Este resultado, contudo, ocorre em função da
hipótese de uma pequena economia aberta, na qual a taxa de juros doméstica é dada pelo mercado
financeiro internacional. 3.4. Simulação 4: Redução do imposto sobre a renda compensada pela introdução
de um imposto lump-sum Da mesma forma que na Sim-2, neste caso não se constata o efeito inicial sobre os preços. Por outro
lado, tanto a alteração do imposto de renda quanto à introdução de um imposto lump-sum, afetam
a renda familiar. A redução do imposto sobre a renda acarreta a expansão da demanda de consumo
das famílias, tanto dos ativos quanto dos inativos. No caso dos ativos, em virtude dos efeitos renda
e substituição, e, no caso dos inativos, somente em função do segundo efeito. Em comparação com a
Sim-3, esta política gera uma maior expansão da demanda de consumo das famílias. Tal fato decorre,
pelos motivos já comentados, das magnitudes dos efeitos renda e substituição. A produção global também se expande mais que no caso precedente. Isto porque, além de estimular
o setor exportador, o maior aumento do consumo interno intensifica a produção doméstica. Como
resultado, tem-se um maior crescimento do PIB e do emprego do que na simulação anterior. Como reflexo do desempenho do setor exportador, verifica-se, à semelhança da simulação anterior,
que tanto o déficit comercial quanto o estoque da dívida externa apresentam substancial redução. A
dívida interna, por sua vez, permanece estável como conseqüência da restrição imposta à manutenção
da receita fiscal estável. empregos para o setor exportador. A expansão das exportações, juntamente com a queda da demanda
de importações, permite uma melhora da balança comercial e a redução da dívida externa. empregos para o setor exportador. A expansão das exportações, juntamente com a queda da demanda
de importações, permite uma melhora da balança comercial e a redução da dívida externa. O efeito da política sobre o bem-estar apresenta-se como negativo, o que revela não parecer ser
desejável a substituição de um imposto indireto sobre oconsumo por um imposto direto sobre a renda. Tal evidência confirma tanto os resultados apresentados por Schubert and Letournel (1991) quanto os
apresentados por Auerbach and Kotlikoff (1987). 4. COMENTÁRIOS FINAIS Utilizando-se de uma estrutura teórica de equilíbrio geral intertemporal, o modelo apresentado
neste artigo permitiu uma análise dos efeitos de longo prazo sobre as variáveis econômicas oriundos
de simulações envolvendo alterações na política tributária. Apesar de ser um modelo relativamente
simples, ele evidenciou alguns aspectos importantes relacionados a este tipo de política. O mais rele-
vante é o de que não é recomendável a substituição de um imposto indireto sobre o consumo por um
imposto direto sobre a renda, mantendo o mesmo nível de receita pública. Tal evidência confirma os
ensinamentos da teoria pertinente, mas vai contra os resultados obtidos em outros estudos, utilizando
modelos estáticos.10 Nas alternativas tributárias que representam uma redução da carga tributária, Sim-1 e Sim-2, verificou-
se uma expansão do produto e do emprego. Em ambas, o consumo interno aumentou o que, devido
ao pleno uso dos fatores produtivos, ocasionou um deslocamento de recursos para o setor produtor
de bens destinados ao mercado doméstico. Como conseqüência, nos dois casos ocorreu um aumento
do déficit comercial e do endividamento externo. O aumento da arrecadação com a expansão da ativi-
dade econômica não compensou a redução de receita com a redução dos impostos e, com isso, a dívida
pública global aumentou. A substituição de impostos diretos e indiretos por um imposto neutro ou lump-sum (Sim-3 e Sim-4)
também mostraram resultados favoráveis sobre o produto e o emprego, embora, como era de se esperar,
com impactos menos significativos que as políticas de redução de impostos.11 Nestes casos, a menor
expansão do consumo fez com que o setor exportador apresentasse um desempenho relativamente
melhor. Como resultado, o saldo comercial melhorou e a dívida externa diminuiu. A dívida interna, por
seu lado, permaneceu estável pela constância da taxa de juros e da receita tributária. Por fim, a redução do imposto sobre o valor agregado, compensada pelo aumento do imposto de
renda (Sim-5), mostrou efeitos desfavoráveis sobre o produto e o emprego. O consumo doméstico se
contrai, levando a uma realocação de recursos para o setor exportador. A expansão das exportações,
juntamente com a queda da demanda de importação, permite uma melhora da balança comercial e a
redução da dívida externa. 10É o caso, por exemplo, de Fochezatto (2003). Neste estudo, o autor trabalha com vários setores e grupos de famílias, capturando
as interligações entre incidência tributária, os diferentes perfis de consumo e a estrutura produtiva setorial. Isto explica as
diferenças de seus resultados com os obtidos neste trabalho. Ando, A. & Modigliani, F. (1963). The “life cycle” hypothesis of saving: Aggregate implications and tests.
American Economic Review, 53(1):55–84. 11Isto, de certa forma, mostra que os atuais impostos, diretos e indiretos, são ineficientes. Ou seja, para uma mesma receita, seus
custos econômicos são maiores que o imposto neutro. Afonso, J. R., Araújo, E. A., Rezende, F., & Varsano, R. (2000). A tributação brasileira e o novo ambiente
econômico: A reforma tributária inevitável e urgente. Revista do BNDES, 7(13):137–170. 3.5. Simulação 5: Redução do imposto sobre o valor agregado compensada pelo
aumento do imposto sobre a renda Esta simulação integra os efeitos das duas últimas simulações. Dado o tamanho relativo das bases
de incidência tributária do consumo e da renda na economia brasileira, a magnitude de elevação da
alíquota do imposto de renda, necessária para manter a receita tributária constante, é maior do que a
sobre o consumo. Como conseqüência, tem-se que o efeito positivo sobre o consumo, provocado pela
redução do imposto sobre o valor agregado, acaba por ser mais do que compensado pelo efeito negativo
devido ao aumento do imposto sobre a renda. O efeito negativo sobre o consumo ocorre tanto na
população ativa quanto na inativa. Observa-se que o PIB, a produção, o emprego e o investimento diminuem em resposta à contração
da demanda das famílias. Mas, se o nível de atividade das empresas que produzem para o mercado
interno se contrai, o mesmo não acontece com aquelas que exportam, ocorrendo uma transferência de 312 RBE
Rio de Janeiro
v. 63 n. 3 / p. 299–314
Jul-Set 2009 Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas Avaliando os Impactos de Políticas Tributárias sobre a Economia Brasileira
com Base em um Modelo de Equilíbrio Geral de Gerações Sobrepostas BIBLIOGRAPHY Afonso, J. R., Araújo, E. A., Rezende, F., & Varsano, R. (2000). A tributação brasileira e o novo ambiente
econômico: A reforma tributária inevitável e urgente. Revista do BNDES, 7(13):137–170. Ando, A. & Modigliani, F. (1963). The “life cycle” hypothesis of saving: Aggregate implications and tests. American Economic Review, 53(1):55–84. 313 RBE
Rio de Janeiro
v. 63 n. 3 / p. 299–314
Jul-Set 2009 Carlos Renato Salami e Adelar Fochezatto Auerbach, A. J. & Kotlikoff, L. J. (1987). Dynamic Fiscal Policy. Cambridge University Press, Cambridge. Ferreira, P. C. G. & Araújo, C. H. V. (1999). Reforma tributária, efeitos alocativos e impactos de bem-estar. Revista Brasileira de Economia, 53(2):133–166. Fochezatto, A. (2003). Reforma tributária, crescimento e distribuição de renda no Brasil: Lições de um
modelo de equilíbrio geral computável. Revista de Economia Aplicada, 7(1):83–110. Lledo, V. D. (2001). Tax reform under fiscal stress: A CGE analysis of the Brazilian tax reform. Disponível
na internet: http://epge.fgv.br/portal/arquivo/1070.pdf. Mercenier, J. & Sampaio de Souza, M. C. (1994). Structural adjustment and growth in a highly indebted
market economy: Brazil. In Mercenier, J. & Srinivisan, T. N., editors, Applied General Equilibrium and
Economic Development. University of Michigan Press, Ann Harbor. Modigliani, F. & Brumberg, R. (1954). Utility analysis and consumption function: An interpretation of
cross-section data. In Kurithara, K., editor, Post-Keynesian Economics. Rutgers University Press, New
Brunswick. Oliveira, L. G. S. & Lannes Jr, O. P. (2000). Reforma da Previdência Social com desequilíbrio orçamen-
tário no regime de repartição: Uma análise de equilíbrio geral com restrições ao crédito. Brasília:
Universidade de Brasília, Seminário 11/00. Schubert, K. & Letournel, P. (1991). Un modèle d’equilibre général appliqué à l’etude de la fiscalité
française: Résultants de long terme. Economie et Prévision, 98:83–99. Tourinho, O. A. F., Kume, H., & Pedroso, A. C. S. (2002). Elasticidade de Armington para o Brasil. Texto
para Discussão 901. Rio de Janeiro: BNDES. 314 RBE
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v. 63 n. 3 / p. 299–314
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Gene drive escape from resistance depends on mechanism and ecology
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bioRxiv (Cold Spring Harbor Laboratory)
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Introduction
1 resistance be genetically unlinked from the drive (Gomulkiewicz
22
et al. 2021). 23 Synthetic gene drives are poised to have profound effects on the
way humans control pests and pathogens. Not only can they be
used to quickly transform wild populations with genes of social
value, even if detrimental to the species, but they can be used
to suppress and even extinguish populations (Hamilton 1967;
Lyttle 1977). The extinction potential has been known for over
half a century, with the remaining hurdle to implementation
having been engineering (Burt 2003). CRISPR has solved that
hurdle, and there are now countless examples in which gene
drives have been genetically engineered, tested, and shown to
work in laboratory populations of selected insects (Bier 2021). .
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bioRxiv preprint Gene drive escape from resistance depends on
mechanism and ecology Forest Cook1, James J. Bull2, ∗and Richard Gomulkiewicz3, ∗
1School of Electrical Engineering & Computer Science, Washington State University, Pullman, WA 99164, 2Dept of Biological Sciences, University of Idaho,
Moscow, ID 83843, 3School of Biological Sciences, Washington State University, Pullman, WA 99164 Forest Cook1, James J. Bull2, ∗and Richard Gomulkiewicz3, ∗
omputer Science, Washington State University, Pullman, WA 99164, 2Dept of Biological Sciences, University of Idaho,
Moscow, ID 83843, 3School of Biological Sciences, Washington State University, Pullman, WA 99164 Forest Cook1, James J. Bull2,
and Richard Gomulkiewicz3,
1School of Electrical Engineering & Computer Science, Washington State University, Pullman, WA 99164, 2Dept of Biological Sciences, University of Idaho,
Moscow, ID 83843, 3School of Biological Sciences, Washington State University, Pullman, WA 99164 ABSTRACT Gene drives can potentially be used to suppress pest populations, and the advent of CRISPR technology has
made it feasible to engineer them in many species, especially insects. What remains largely unknown for implementations is
whether anti-drive resistance will evolve to block the population suppression. An especially serious threat to some kinds of drive
is mutations in the CRISPR cleavage sequence that block the action of CRISPR, but designs have been proposed to avoid
this type of resistance. Various types of resistance at loci away from the cleavage site remain a possibility, which is the focus
here. It is known that modest-effect suppression drives can essentially ‘outrun’ unlinked resistance even when that resistance is
present from the start. We demonstrate here how the risk of evolving (unlinked) resistance can be further reduced without
compromising overall suppression by introducing multiple suppression drives or by designing drives with specific ecological
effects. However, we show that even modest-effect suppression drives remain vulnerable to the evolution of extreme levels
of inbreeding, which halt the spread of the drive without actually interfering with its mechanism. The landscape of resistance
evolution against suppression drives is therefore complex, but avenues exist for enhancing gene drive success. 1
2
3
4
5
6
7
8
9
10
11
KEYWORDS gene drive; non-allelic resistance; CRISPR; population suppression; homing drive; toxin-antidote drive
12 KEYWORDS gene drive; non-allelic resistance; CRISPR; population suppression; homing drive; toxin-antidote drive ∗Corresponding authors: gomulki@wsu.edu, jbull@uidaho.edu .
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bioRxiv preprint Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 It was previously shown that moderate-effect suppression drives
117
could evolve to fixation even when resistance was initially
118
present. The drive’s fitness effect was fully recessive, mani-
119
fest only in drive homozygotes, and segregation distortion was
120
100%. Furthermore, and unexpected, suppression drives could
121
evade resistance even if there was no fitness cost to the resistance
122
allele (Gomulkiewicz et al. 2021); those models assumed type-M
123
resistance. 124 With type-M resistance against a homing drive, there is the
possibility of non-functional resistance – target-site mutations
that block the drive and destroy the target-site gene function. Non-functional resistance can affect the spread and long-term
impact (Beaghton et al. 2019). Non-functional resistance will be
ignored here. In that prior study, there were limits on the suppressive effect
of the drive that could evade resistance. The maximum possi-
ble suppression decreased with linkage between the resistance
and drive loci, decreased with initial frequency of the resistance
allele, but the possible suppression was considerably increased
if the drive’s fitness effect was manifest in only one sex (Go-
mulkiewicz et al. 2021). Two-sex drives were only slightly better
at evading cost-free resistance than one-sex drives when drive
fitness effects were experienced in both sexes. (To clarify, ‘one-
sex’ and ’two-sex’ drives refer to the drive’s selfish advantage
operating in one sex, such as only males, or both sexes.) Here we
report that the fitness suppression attainable from ’resistance-
proof’ drives can be substantially improved when introducing
2 drives whose fitness effects combine multiplicatively. The in-
troduction of multiple, simultaneous suppression drives was in
fact proposed by Burt (2003) as a means to force a population
to low numbers before resistance could ascend; our emphasis
is somewhat different in that we consider drives that can fix in
the presence of resistance. We confine this part of our analy-
sis to type-M resistance and drives that operate in males only
(one-sex drives), as that is a slightly more difficult case for evad-
ing resistance. Male drives have the advantage of avoiding
Cas9 carryover to and unwanted nuclease activity in the embryo
(Champer et al. 2017). Our study assumes that resistance is unlinked to the drive. Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 This assumption is not typical of other studies – most analyses
of resistance to a gene drive have assumed that resistance is
allelic to the drive (e.g., Unckless et al. 2015, 2017; Beaghton et al. 2017; Noble et al. 2017; Prowse et al. 2017; KaramiNejadRanjbar
et al. 2018), although for an exception see (Charlesworth and
Hartl 1978). Allelic resistance is highly relevant to a homing
endonuclease for which resistance may take the form of a mu-
tation in the target site (Champer et al. 2017; Noble et al. 2017;
Bier 2021; Hammond et al. 2021; Terradas et al. 2021) as well
as to some toxin-antidote systems, but there are designs that
may avoid allelic resistance (see Discussion). Many other forms
of resistance will not be allelic to the drive allele and not in-
volve mutations at the cleavage site, such as blocks to gene drive
expression, interference with endonuclease protein complexes,
inbreeding, and others. The evolution of non-allelic resistance
is fundamentally different from allelic resistance because the
benefit of resistance no longer accrues directly to the resistance
allele. The genetic separation of drive and resistance weakens
selection of resistance and allows some forms of suppression
drives to escape resistance evolution (Gomulkiewicz et al. 2021). The model has 3 unlinked loci, each with 2 alleles: A/a
(homing drive 1), B/b (homing drive 2), and R/r (resistance),
lowercase alleles representing wild-type; resistance acts equally
against both drives. Other assumptions about segregation distor-
tion and fitness effects are provided in Tables 1 and 2. Complete
segregation distortion operates independently at the A/a and
B/b loci; dominant, cost-free resistance to segregation distortion
operates at the R/r locus (Table 1). We address avoidance of resistance evolution in several di-
verse contexts. The cost of such diversity is that each case can be
explored in only some of the vast diversity of possible variations
that are of interest: drive imperfection, fitness effects of different
genotypes, dominance/recessivity within loci, deviations from
random mating, and ecology. To keep the study manageable,
most of our models assume perfect drive with fitness effects
in only the drive homozygote and no fitness cost to resistance:
these are extremes that represent best cases for the evolution of
resistance (no fitness cost) with a drive that has the best chance
of outrunning resistance. The present study builds on our previ-
ous study that explored some of these variations (Gomulkiewicz
et al. 2021). Realms of resistance In understanding gene drive evasion of resistance evolution, it
25
is necessary to contemplate the different types of resistance that
26
may occur, because the type of resistance affects the evolutionary
27
dynamics. We can identify three distinct types of resistance (see
28
also Bull et al. 2019)):
29 ‘M’ Mechanistic. The drive mechanism is blocked in the in-
30
dividual, impairing the drive’s selfish advantage. With
31
CRISPR, this could take the form of a mutation at the en-
32
donuclease cleavage site that blocks cutting or a protein
33
that interferes with CRISPR function (Taxiarchi et al. 2021;
34
Jia and Patel 2021). Some of these would necessarily be
35
allelic to the drive, others could be unlinked. 36 y p p
What remains is to understand whether suppression drives
can escape resistance evolution. The fitness consequences of a
suppression drive create an advantage for genes that block the
drive, and if those blocks evolve, the suppression drive can be
rendered ineffective (Lyttle 1979, 1981). If a suppression drive
is to have a lasting effect, especially cause extinction, it either
needs to be designed so that resistance is mutationally unlikely
or not tolerated (Kyrou et al. 2018; Champer et al. 2018) or that it
evolves to fixation ahead of resistance – which requires that the ‘C’ Compensatory. The drive evolves normally but the fitness
3
consequence of the drive is abrogated by evolution at an-
3
other site, such as compensatory evolution elsewhere in the
3
genome (Lyttle 1981; Burt 2003). 4 g
y
‘P’ Population structure. The population develops a structure
41
that enables family or group selection to keep the drive
42 . CC-BY 4.0 International license
available under a
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bioRxiv preprint . CC-BY 4.0 International license
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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bioRxiv preprint Models and Results from spreading/fixing (Bull 2016; Bull et al. 2019). The
population structure need not evolve per se but may instead
arise dynamically as the population size declines (Bull 2016;
Bull et al. 2019; North et al. 2019; Champer et al. 2021). Our methods use mostly numerical analyses of deterministic,
discrete-time gene frequency models; the ecology section ex-
tends the models to population dynamics (code access is ad-
dressed in the Data availability statement). Mating is random
unless specified otherwise. Two or three unlinked loci are as-
sumed, each with 2 alleles. Some models assume that the sexes
are haploids, other models assume diploid sexes. Computation
employed C programs with graphics in R (R Core Team 2019). The practicality of avoiding these alternative resistances will
depend on the application as well as the engineering. The hope
is that it will be possible to design drives that are a priori prone
to escape many forms of resistance evolution. That is our thesis
here. We expand on earlier work showing it was possible to
develop moderate-effect suppression drives that would ’out-run’
unlinked mechanism-blocking resistance (type-M) already exist-
ing in the population (Gomulkiewicz et al. 2021). But there were
limits on how much population suppression could be achieved
while evading resistance. We find here that ways exist to allow
even greater magnitudes of population suppression while evad-
ing resistance evolution. Critically, this resistance must be at
least partly unlinked to the drive, because the drive allele cannot
otherwise fix (deterministically). However, we also show that
this principle for evading type-M resistance does not translate
equally to other resistance types. We also explore resistance
evolution in the context of ecology. Cook et al. 2
Cook et al. Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 If only a single drive is intro-
duced, s is the effect of the one drive (its homozygote fitness
is 1 −s). For each initial frequency of resistance, the maxi-
mum resistance-free s was obtained over 100 trials spanning
different levels of gene drive fitness effects (incremented ev-
ery 0.01). The main point of the graph is that two suppression
drives (blue) can escape resistance evolution and attain greater
combined population suppression than can a single drive (yel-
low) of the same total effect; the advantage of 2 drives applies
whether the fitness effects are experienced by both sexes (solid
blue versus solid yellow) or just females (dashed blue versus
dashed yellow), but a larger s can evolve resistance-free if the
fitness effect is confined to females (dashed versus solid of
same color). There is essentially no difference whether the two
drives are introduced concurrently or sequentially, so sequen-
tial drive outcomes are shown. These suppression maxima
were enduring, the drive allele(s) having fixed despite resis-
tance being present in the population. The resistance allele
persists but has no effect once the drive is fixed. The horizon-
tal axis is the initial frequency of the (dominant) resistance
allele, and it is easily seen that greater resistance-free suppres-
sion depends on a lower starting frequency of the resistance
allele. Initial frequencies of the drive alleles were 0.005 when
two drives were present, 0.01 for a single drive; some trials
used initial frequencies of 0.0005, with no appreciable change
from results with initial frequencies of 0.005. The leftmost ini-
tial frequency of the resistance allele was 0.005, incremented
by 0.01. Table 2 Diploid genotype fitnesses for the 3-locus model with
two homing drives and a resistance locus. A dash (–) indi-
cates either allele at the locus. Drive alleles are A and B, resis-
tance is R. The parameter s determines the fitness reduction in
drive homozygotes; fitnesses are multiplicative over loci. All
genotypes not shown have fitness 1. Fitness effects operate on
adults during gamete production. In some models, the fitness
effects apply to both sexes, in others just to females, as given
in Fig. 1. Cost-free resistance is assumed, as it is the most fa-
vorable case for resistance evolution. If a drive can evade cost-
free resistance, then it should also evolve in all cases of costly
resistance. Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 If only a single drive is intro-
duced, s is the effect of the one drive (its homozygote fitness
is 1 −s). For each initial frequency of resistance, the maxi-
mum resistance-free s was obtained over 100 trials spanning
different levels of gene drive fitness effects (incremented ev-
ery 0.01). The main point of the graph is that two suppression
drives (blue) can escape resistance evolution and attain greater
combined population suppression than can a single drive (yel-
low) of the same total effect; the advantage of 2 drives applies
whether the fitness effects are experienced by both sexes (solid
blue versus solid yellow) or just females (dashed blue versus
dashed yellow), but a larger s can evolve resistance-free if the
fitness effect is confined to females (dashed versus solid of
same color). There is essentially no difference whether the two
drives are introduced concurrently or sequentially, so sequen-
tial drive outcomes are shown. These suppression maxima
were enduring, the drive allele(s) having fixed despite resis-
tance being present in the population. The resistance allele
persists but has no effect once the drive is fixed. The horizon-
tal axis is the initial frequency of the (dominant) resistance
allele, and it is easily seen that greater resistance-free suppres-
sion depends on a lower starting frequency of the resistance
allele. Initial frequencies of the drive alleles were 0.005 when
two drives were present, 0.01 for a single drive; some trials
used initial frequencies of 0.0005, with no appreciable change
from results with initial frequencies of 0.005. The leftmost ini-
tial frequency of the resistance allele was 0.005, incremented
by 0.01. 0.0
0.1
0.2
0.3
0.4
0.0
0.2
0.4
0.6
0.8
Initial frequency of resistance
max resistance-proof s
Drive type
two drives
two drives f only
one drive
one drive f only Genotype
Fitness
aa
bb
rr
1
AA
b –
– –
√
1 −s
a –
BB
– –
√
1 −s
AA
BB
– –
1 −s Figure 1 Maximum fitness suppression attainable with 1 ver-
sus 2 homing drives evolving in the presence of strong, cost-
free resistance alleles; drive operates in males only. Curve
height is the maximum fitness suppression attainable (s). When two drives are introduced, s is the combined effect
of both drives, from Table 2. Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 The application of principles found here to any
implementation will require models specific to that case. Figure 1 compares the maximum s attainable by one drive
versus 2 drives while evading cost-free resistance. These curves
apply to drive alleles that evolve to fixation, after which type-
M resistance can no longer influence evolution or fitness. The
extreme case of resistance-free suppression (70%) is attained
using 2 drives with equal fitness effects confined to just females. These figures assume that the two drives are introduced sequen-
tially (second drive introduced when first drive has attained a
frequency of 0.9995, dashed curves), but essentially the same 2 . CC-BY 4.0 International license
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bioRxiv preprint Male genotype
Gametes produced
Aa Bb rr
ABr
Aa bb rr
Abr
aa Bb rr
aBr Table 1 Gamete production for the three genotypes that are
heterozygous at drive loci and wild-type homozygous at
the resitance locus. Drive operates in males only via hom-
ing. Segregation is Mendelian for the 24 other possible male
diploid genotypes not shown here and for all 27 possible fe-
male diploid genotypes. Resistance to segregation distortion
is complete and dominant, and the R resistance allele fully
blocks drive by A and B, separately and together. 0.0
0.1
0.2
0.3
0.4
0.0
0.2
0.4
0.6
0.8
Initial frequency of resistance
max resistance-proof s
Drive type
two drives
two drives f only
one drive
one drive f only
Figure 1 Maximum fitness suppression attainable with 1 ver-
sus 2 homing drives evolving in the presence of strong, cost-
free resistance alleles; drive operates in males only. Curve
height is the maximum fitness suppression attainable (s). When two drives are introduced, s is the combined effect
of both drives, from Table 2. .
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bioRxiv preprint Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 Since the diploid phase is
247
neither male nor female, this model is effectively a 2-sex homing
248
drive. There is no genetic resistance to the drive action as such,
249
as all drive heterozygotes produce only A gametes (Table 3). 250 free suppression by 2 drives: to attain a combined suppression
185
of 1-s, the selective coefficient per drive needed when using
186
2 drives is slightly more than s/2, the excess increasing with
187
s. The relative gain from using 2 drives is thus due to the net
188
balance of these three components. A theoretical possibility
189
is that arbitrary resistance-free population suppression could
190
be obtained by using many drives of very small effect, but the
191
method is not obviously practical beyond two or three drives. 192 Diploid genotype
Diploid fitness
Progeny produced Diploid genotype
Diploid fitness
Progeny produced
aa
1
a
Aa
1
A (after drive)
AA
1 −s
A y p
y
Robustness. The model assumed a few extremes: perfect hom-
ing drives and no fitness costs to drive heterozygotes. To address
the importance of the first extreme, trials were analyzed with
drive distortion of 0.9 (drive heterozygotes produce 90% drive
and 10% wild type gametes); this analysis was conducted for
two male drives introduced simultaneously with equal fitness
effects in both sexes and compared to that of a single male drive
(corresponding to the solid lines in Fig. 1). There was little
effect of this level of drive imperfection: the difference in max-
imum resistance-free s between a single drive and two drives
changed less than 0.01 when comparing perfect drives to im-
perfect drives. A second check on robustness was to introduce
a fitness decrement in drive heterozygotes. Surprisingly, a re-
duction in heterozygote fitness of 0.1 (from 1.0 to 0.9) increased
the maximum resistance-free s. For perfect drive, the gain in
resistance-free s was substantial even for a single drive (e.g.,
0.06) and often somewhat larger for two drives (up to 0.09). For
imperfect drives (90%, 10%, as above), the gain in resistance-free
s by a 0.1 reduction in heterozygote fitness could be somewhat
larger or smaller than the gain for perfect drives. The main
observation remains that the use of 2 drives can attain a larger
net suppression than the use of a single drive despite moderate
variations in drive perfection and heterozygote fitness. Inbreeding can evolve to block homing drives of even modest
effect: 2-sex drives Table 4 Relationship between maternal genotype at the Q/q lo-
cus and the fraction of her progeny that engage in sib mating. Table 4 Relationship between maternal genotype at the Q/q lo-
cus and the fraction of her progeny that engage in sib mating. The models both above and in previous work (Gomulkiewicz
21
et al. 2021) assumed type-M resistance, thus blocking segrega-
22
tion distortion in the individual heterozygous for the drive(s). 23
Resistance evolves because of a statistical association between
24
the resistance allele and the drive-sensitive allele(s). Type-P
25
resistance (population structuring) offers a different way to de-
26
velop a statistical association between drive-sensitive alleles and
27
resistance. We thus consider whether type-P resistance behaves
28
similarly as type-M resistance in allowing modest-effect drives
29
to fix without selecting resistance. 30 It is further assumed that sib mating does not affect offspring
259
fitness (no inbreeding depression). This assumption is equiv-
260
alent to cost-free type-M resistance and enables isolating the
261
drive-blocking effect of sib mating. It is obviously a best-case
262
scenario for the evolution of type-P resistance, and although it
263
may be unrealistic, it should also define the conditions under
264
which a suppression drive can be assured of evading sib mating. 265
266 Figure 2 shows three scenarios differing in the degree of sib
267
mating imposed by mothers with allele Q, from low levels of
268
sib mating (m = 0.2), medium (m = 0.5) to high levels (m =
269
0.95). Each graph displays equilibrium mean fitness (average
270
female survival), equilibrium drive frequency, and equilibrium
271
frequency of Q for different values of drive fitnesses (1 −s); (note
272
that sib mating has no fitness consequences if and after the drive
273
fixes, so polymorphism of Q after drive fixation merely reflects
274
its spread prior to drive fixation). There are three important
275
outcomes. 276 g
To represent type-P resistance, we invoke sib mating (Bull
et al. 2019). Sib mating is not resistance in the sense of block-
ing segregation distortion, indeed it has no effect on distor-
tion. Rather it provides a population structure in which drives
are disproportionately partitioned across groups of individuals,
thereby increasing the variance in fitness across families. Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 Note that fitness of a genotype translates into pop-
ulation fitness to the extent that the genoytpe comprises the
population. curves are obtained for simultaneous introduction, suggesting
that the major benefit of two drives is not from some interaction
between the combination of two drive alleles and the resistance
allele. The greater impact of resistance-proof suppression by using
170
2 drives instead of a single drive begs some understanding. 171
We addressed this question with numerical results using our
172
observation that 2 sequential drives can attain approximately
173
the same total suppression as 2 concurrent drives; properties
174
of gene frequency evolution were analyzed during single drive
175
fixation (confining the analyses to parameter values for which
176
the drives evolved to fixation). We found that two outcomes
177
work in favor of greater resistance-free suppression by 2 weak
178
drives than by 1 stronger drive. (1) For the same initial frequency
179
of the resistance allele, larger values of s disproportionately lead
180
to higher final frequencies of the resistance allele. (2) For the
181
same value of s, higher initial frequencies of the resistance allele
182
yield higher gains of the resistance allele (only noticeable at
183
larger values of s). A third outcome works against resistance-
184 Resistance proofing gene drives 3 . CC-BY 4.0 International license
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bioRxiv preprint only if the diploid is Aa, and fitness effects of drive occur if and
246
only if the diploid is AA (Table 3). Two homing drives can escape resistance while achieving
114
greater total suppression than does a single drive:
1-sex
115
drives
116 It is of
course understood that drive properties differing between the
2-drive and 1-drive cases could affect the benefit of using two
drives. Table 3 Gamete production and fitnesses for the three drive
genotypes in the sib-mating model. Drive operates in Aa
diploids only and does not affect other loci. Drive is not sup-
pressed or affected by other loci. These properties apply to
all diploids. Diploids are not distinguished as male or female,
as sexes apply to haploids. s is manifested as viability of AA
progeny. Sib mating is enforced by the mother on her progeny (Table 4). 251
Only mothers of genotype Q have sib-mated progeny, and then
252
only a fraction m of those sib mate. All offspring of q mothers
253
and 1 −m offspring of Q mothers join the random mating pool. 254
We assume that males who mate with their sisters do not also join
255
the random pool, which would give sib mating an advantage
256
independent of gene drives (Lande and Schemske 1985). The
257
fitness effect 1 −s is manifested as viability of AA progeny. 258 4
Cook et al. 4
Cook et al. Inbreeding can evolve to block homing drives of even modest
effect: 2-sex drives In
addition, the inbreeding itself reduces the frequency of drive
heterozygotes, which are the only genotypes that permit the
drive to collect its ‘unfair’ transmission advantage. In contrast
to previous sib-mating models (Bull et al. 2019), we here allow
drive homozygotes to be partly viable – a necessary condition for
type-M drives to evade low levels of pre-existing resistance and
thus an appropriate parallel for comparison. To keep the math
simple, we assume sexual haploids: mated females produce a
brief diploid stage in which segregation distortion occurs if and 1) Sib mating does not evolve (and the drive evolves to fixa-
27
tion) unless the sib-mating fraction exceeds the fitness of
27
the drive homozygote (m > 1 −s). Thus, mild-effect drives
27
can avoid type-P resistance evolution if there are only low
28
levels of sib mating types in the population. 28 . CC-BY 4.0 International license
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bioRxiv preprint 2) When it evolves enough to at least partially suppress the
82
drive, the sib-mating allele typically fixes. For some com-
83
binations, the drive allele can be maintained polymorphic
84
despite fixation of allele Q. We have not observed joint
85
polymorphism of Q/q and the drive allele, but we cannot
86
rule it out. 87 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
A. sib mating rate m = 0.2
drive selection coefficient s
mean fitness/frequency
type M resistance
drive allele frequency
sib mating allele frequency
mean fit. with sib mating
mean fit. random mating
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
B. sib mating rate m = 0.5
drive selection coefficient s
mean fitness/frequency
type M resistance
drive allele frequency
sib mating allele frequency
mean fit. with sib mating
mean fit. Inbreeding can evolve to block homing drives of even modest
effect: 2-sex drives random mating
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
C. sib mating rate m = 0.95
drive selection coefficient s
mean fitness/frequency
type M resistance
drive allele frequency
sib mating allele frequency
mean fit. with sib mating
mean fit. random mating
Figure 2 (Caption on next column.) A. sib mating rate m = 0.2 3) If the sib-mating genotype enforces a high degree of sib
mating (m near 1), sib mating evolves to extinguish all but
the most benign gene drives. 3) If the sib-mating genotype enforces a high degree of sib
mating (m near 1), sib mating evolves to extinguish all but
the most benign gene drives. 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
drive selection coefficient s
mean fitness/frequency
type M resistance
drive allele frequency
sib mating allele frequency
mean fit. with sib mating
mean fit. random mating In comparison to homing drive evasion of type-M resistance,
291
sib mating provides a mixed message. Modest effect drives
292
can fix if the population lacks alleles that enforce high levels of
293
sib mating, even if the population has alleles that enforce low
294
levels of sib mating (Fig. 2A, B). This offers a parallel to modest-
295
effect drives evading type-M resistance whereas lethal drives
296
cannot evade resistance. In contrast, a recessive lethal drive
297
(s = 1) will select any level of sib mating (all m > 0), although
298
the equilibrium frequency of the drive remains high if the sib
299
mating allele enforces only low levels of sib mating (Fig. 2A). 300 Seemingly without parallel in type-M resistance, homing
drives of moderate or even small effect cannot evolve to fixation
if the population has genotypes that enforce high levels of sib
mating (Fig. 2C). In this sense, sib mating provides a graded
resistance response – if the population has even a low frequency
of genotypes that impose high levels of sib mating, they will
evolve to stop even weak-effect drives. But if the population
has only genotypes that impose low levels of sib mating, even
moderate-effect drives can evolve to fixation. Unfortunately, it
may be difficult to anticipate in a wild population what levels of
genetically controlled sib mating exist at low frequency. 0.2
0.4
0.6
0.8
drive selection coefficient s 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
B. Inbreeding can evolve to block homing drives of even modest
effect: 2-sex drives sib mating rate m = 0.5
drive selection coefficient s
mean fitness/frequency
type M resistance
drive allele frequency
sib mating allele frequency
mean fit. with sib mating
mean fit. random mating B. sib mating rate m = 0.5 There is an interesting difference in drive evolution between
the m = 0.95 case and the other two cases considered in Fig. 2. When the m = 0.95 sib mating allele evolves to fixation, the
drive allele is purged. In contrast, for m = 0.2 and m = 0.5, the
drive remains polymorphic or even abundant despite fixation
of the sib-mating allele. Persistent drive polymorphism could
select additional types of resistance. Robustness. These results were based on perfect drive and no
heterozygote fitness effect of the drive. Revisiting the three cases
in Fig. 2, those assumptions were relaxed by allowing a moder-
ately imperfect drive (drive heterozygotes produce 90% drive
and 10% wild type gametes) and/or a heterozygote fitness cost
of 0.1. The effects were quantitative and did not alter the overall
outcomes. Drive imperfection reduced the range of s values in
which drive fixed by 0.01 for m = 0.95 to 0.05 for m = 0.2. Mean
fitness in the zone of drive polymorphism increased substan-
tially with drive imperfection alone for m = 0.5 and m = 0.2 (by
as much as 0.18 and 0.26, respectively), with the heterozygote
fitness cost also increasing mean fitness but less so. The basic
result that high magnitudes of sib mating purge drives of even
small s effect remains. The fact that drive imperfection reduces
population suppression in the zones of drive polymorphism
is not surprising, and the magnitude of effect may have little
permanance given that the polymorphic drive would be prone
to select other forms of resistance. 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
C. sib mating rate m = 0.95
drive selection coefficient s
mean fitness/frequency
type M resistance
drive allele frequency
sib mating allele frequency
mean fit. with sib mating
mean fit. random mating C. sib mating rate m = 0.95 Figure 2 (Caption on next column.) Sib mating can evolve in response to toxin-antidote drives, but
337
patterns differ from homing drives
338 The
orange curve gives evolved mean fitness (average female sur-
vival, since male frequencies were normalized to 1); the green
curve gives equilibrium fitness in the absence of sib mating;
the pink curve gives the frequency of the sib-mating allele
Q. Panels differ in the level of sib mating, m, imposed by the
sib-mating allele Q: 0.2 (panel A), 0.5 (panel B), 0.95 (panel
C). Analytical results (supplemental file S1) show that the
gene drive fixes if selection against it is below the level of out-
crossing enforced by the inbreeding allele (i.e., if s < 1 −m)
and is lost if s > 1/m −1; at extremes of m, the drive allele is
vanquished. The numerical results agree with that behavior. For 1 −m < s < min(1/m −1, 1), polymorphic drive equilibria
exist with allele frequency equal to 1/s −m/(1 −m). Overall,
Q alleles with high values of m evolve to fixation at all but low
values of s, but whatever the value of m(> 0), Q will evolve to
fixation as s gets high enough. But once a value of s is reached
such that Q fixes, if the drive allele remains polymorphic, in-
creased values of s lead to lower drive frequency and higher
mean fitness. For comparison, the shaded area shows the val-
ues of s for which an unlinked, type-M resistance allele would
evolve to block the drive. The curves in each panel were gen-
erated from trials incremented every 0.01 on the X-axis; initial
frequencies were 0.01 for both the sib-mating allele and the
drive allele (or the resistance allele for type-M resistance). Out-
comes were assessed at 105 generations. Note that in (C), the
orange curve follows the green only at low values of s and
then rises to join the pink throughout most of the range of s. When CC fitness is impaired, ClvR faces the same threat
362
of allelic resistance as do homing drives. A target-site allele g
363
that is resistant to cleavage but retains function will eventually
364
ascend and prevent evolution of C. The same designs used for
365
avoidance of functional allelic resistance in homing drives will
366
thus need to be used for ClvR (see Discussion). Our interest here
367
is in the evolution of sib mating as a form of type-P resistance. Sib mating can evolve in response to toxin-antidote drives, but
337
patterns differ from homing drives
338 Inbreeding has a moderately straightforward interaction with
339
homing drives, which are confined to one locus. If the principle
340
behind the evolution of sib mating in response to a suppression
341
drive is that it increases the variance in fitness across families,
342 Resistance proofing gene drives Resistance proofing gene drives 5 . CC-BY 4.0 International license
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bioRxiv preprint one would expect sib mating to be favored with other forms of
suppression drives. Here we consider evolution of sib mating in
response to toxin-antidote systems used for suppression. There is a large variety of possible toxin-antidote designs
(Champer et al. 2020a). Our example of a toxin-antidote drive is
ClvR (Oberhofer et al. 2019) or equivalently distant-site TARE
(Champer et al. 2020b). Two loci are involved. At the ClvR locus,
with alleles c/C, the C allele encodes both a rescue gene and a
nuclease. The nuclease annihilates wild-type alleles at a second,
unlinked, essential-gene locus, g/G (Table 5). ClvR (allele C) is
engineered as a one-locus construct with two functions, the g/G
locus merely being the passive ‘victim’ of the nuclease of the
first locus. The C allele spreads because it creates non-functional
G alleles, with GG genotypes dying if not rescued by C (ccGG is
the only genotype that dies because it lacks the rescuing C allele). ClvR does not intrinsically suppress populations. To be used
for population suppression, the C can be inserted into a gene
important to survival/fecundity so that the gene is disrupted
and homozygotes impaired (Table 5). Figure 2 Equilibrium consequences of homing drive evolu-
tion when confronted with evolution of population structure
resistance (type-P) in the form of sib mating. The black curve
provides the equilibrium frequency of the drive allele. Sib mating can evolve in response to toxin-antidote drives, but
337
patterns differ from homing drives
338 The equilibrium frequency of C with a fixed in-
breeding coefficient f is ˆpC = (1 −f σ)/[(1 + σ)(1 −f )]. At
least when rare, inbreeding would likely be selected against
as the population neared this state since less inbred families
would produce more descendants. This behavior contrasts
with that of homing drives considered above, which have
no effect on sib mating once they are fixed. 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
A. m = 0.2
drive selection coefficient s
mean fitness
sib mating
random mating
type M resistance
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
B. m = 0.5
drive selection coefficient s
mean fitness
sib mating
random mating
type M resistance
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
C. m = 0.95
drive selection coefficient s
mean fitness
sib mating
random mating
type M resistance
Figure 3 (Caption on next page.) 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
A. m = 0.2
drive selection coefficient s
mean fitness
sib mating
random mating
type M resistance
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
B. m = 0.5
drive selection coefficient s
mean fitness
sib mating
random mating
type M resistance 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
A. m = 0.2
drive selection coefficient s
mean fitness
sib mating
random mating
type M resistance A. m = 0.2 A. m = 0.2 g
y
• Inbreeding affects and is affected by two loci with ClvR: the
c/C locus (as noted above) and the g/G locus, for which
GG is lethal in the absence of C. With homing drives, only
one locus is affected by inbreeding. • As elaborated in the next section, ClvR cannot suppress
392
mean fitness below 0.5 (when fitness effects are confined
393
to the homozygote as viability effects). The effect of fitness
394
parameter σ on mean fitness is not the same as that of the
395
homing drive parameter s. 396 sib mating
random mating These differences raise the possibility that sib mating will evolve
397
differently under ClvR than under homing drives. Sib mating can evolve in response to toxin-antidote drives, but
337
patterns differ from homing drives
338 368
Sib mating operates as in the previous model (Table 4); the
369
model again assumes haploid sexes, but fitness effects and allelic
370
conversion occurs in diploids (Table 5). 371 Genotype
fitness
behavior
cc g –
1
cc GG
0
Cc g –
1
g −→G
Cc GG
1
CC g –
1-σ
g −→G
CC GG
1-σ Genotype
fitness
behavior
cc g –
1
cc GG
0
Cc g –
1
g −→G
Cc GG
1
CC g –
1-σ
g −→G
CC GG
1-σ Table 5 Genotypes, fitnesses and functions of the 2 loci in the
ClvR toxin-antidote system; a dash (-) represents either allele. Allele C performs two functions: it converts all wild-type g
alleles in the individual to non-functional G, and it rescues GG
from death. The fitness parameter here is σ, to distinguish it
from s in homing drive models. These properties apply to all
diploids. Diploids are not distinguished as male or female, as
sexes apply to haploids. Assigning a fitness decrement to Cc
heterozygotes imposes a threshold frequency requirement for
spread of C when rare (Oberhofer et al. 2019; Champer et al. 2020b). In contemplating the evolution of inbreeding, use of ClvR
372
for population suppression exhibits some notable differences
373
from homing drives. Note that we are confining our analysis to
374
viability effects of C on both sexes:
375 • In the absence of resistance, evolution of ClvR leads to a
376
population that is fixed for null homozygotes GG but poly-
377
morphic for C/c: CC has low fitness, Cc is normal, and cc
378 6
Cook et al. Cook et al. 6 6 . CC-BY 4.0 International license
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bioRxiv preprint dies because all wild-type g are gone. This fitness overdom-
inance holds for any σ > 0 and is a basis for inbreeding
depression. Sib mating can evolve in response to toxin-antidote drives, but
337
patterns differ from homing drives
338 398 Plots of sib mating evolution in response to ClvR evolution
99
show similar patterns as for homing drives in that strong allelic
00
sib mating can evolve to block the suppressing ClvR (compare
01
Fig. 3 to 2). There are two differences worth noting, however:
02 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
B. m = 0.5
drive selection coefficient s
mean fitness
sib mating
random mating
type M resistance 1. Alleles enforcing weak sib mating evolve only at higher
403
values of σ than for s with homing drives. For example,
404
with m = 0.5, ClvR does not select sib mating until σ is al-
405
most 0.7, whereas homing drives selected sib mating when
406
s reached 0.5. 407 2. At equilibrium, joint polymorphism of C and sib mating
408
was never observed, although we cannot rule out the possi-
409
bility. If sib mating evolved, ClvR was lost, at which point
410
the Q allele was neutral. Yet with homing drives, the drive
411
allele could be maintained after Q evolved to fixation (for
412
some conditions). 413 These results may not be representative of all outcomes at
414
least because our explorations are limited to one set of initial
415
frequencies. We expect that the interior dynamics of the joint
416
evolution of ClvR and sib mating to be complicated because
417
of the interaction among the 3 loci. Indeed, cursory numerical
418
explorations have observed internal equilibria. The main point
419
is that, as with suppression homing drives, a suppression toxin-
420
antidote system can select inbreeding under a wider range of s
421
than for which type-M resistance evolves. 422 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
C. m 0.95
drive selection coefficient s
mean fitness
sib mating
random mating
type M resistance
Fi
3 (C
ti
t
) Fitness suppression potential across different drive systems
423 Fitness suppression potential across different drive systems
423
The goal of a suppression drive application is to reduce actual
424
population sizes of a species. Analyses above were based on
425
average fitness effects, a genetic measure, and the translation
426
between a reduction in fitness and the demographic effect on
427
population size can be complicated. This section offers a prelude
428
to the study of ecological effects by revisiting how mean fitness
429
reduction depends on the type of drive (Fig 4). 430 Figure 3 (Caption on next page.) In the absence of resistance, a 2-sex homing drive can evolve
431
to suppress mean fitness anywhere from 1.0 to 0 depending
432
on the fitness 1 −s of the drive homozygote (see Prout 1953,
433
for the case of s = 1). In contrast, a 1-sex homing drive can
434
suppress mean fitness no lower than 0.5 (Bruck 1957). When s
435
exceeds 0.5 in a 1-sex homing drive, the equilibrium population
436
is polymorphic for the drive because the non-drive sex produces
437 Resistance proofing gene drives 7 . CC-BY 4.0 International license
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bioRxiv preprint 50% wild-type gametes; mean fitness remains at 0.5 for the entire
range of 0.5 < s < 1. ClvR, our representative toxin-antidote system, has elements
440
of 2-sex and 1-sex homing drives. It operates in both sexes, but
441
it’s equilibrium fitness mirrors that of 1-sex homing drives. Avoiding resistance using ecology: homing drives Many applications of gene drives will be designed for ecological
effects – population suppression or eradication. It is not straight-
forward to translate the relative fitness effect of a gene drive into
ecology: how does a 50% reduction in birth rate translate into
a change in adult population size, for example? Furthermore,
it may be possible to exploit desired ecological effects while
evading evolution of resistance by choosing ecological traits that
amplify effects on population size while reducing relative fitness
effects. One such example is to limit drive fitness consequences
to females, thereby essentially halving the relative fitness effect
but yielding the same impact on population growth as if males
were also affected. 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
drive selection coefficient s or s
mean fitness (in females)
Drive Type
ClvR
two sex homing
one sex homing
Figure 4 Equilibrium mean fitness (average survival) as a func-
tion of the drive homozygote fitness effect for single 1-sex
homing drives (purple), 2-sex homing drives (green), and the
ClvR toxin-antidote drive (blue); s applies to homing drives,
σ to ClvR. These outcomes assume that the fitness effect oper-
ates in both sexes, that drive has evolved to its maximum level,
and that resistance is absent; similar curves apply if the fitness
effect is confined to one sex and mean fitness is measured in
that sex. Only 2-sex homing drives can suppress mean fitness
below 0.5 for these systems, although some toxin-antidote sys-
tems can suppress mean fitness even lower (Champer et al. 2020a). A comparison of fitness effects across different drive
systems is also provided by Beaghton et al. (2019). 0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
drive selection coefficient s or s
mean fitness (in females)
Drive Type
ClvR
two sex homing
one sex homing Although ecological processes collectively offer a one-to-
many translation of relative fitness into adult population size,
depending on assumptions about ecology (e.g., Nagylaki 1992),
we expect that the type of drive will not strongly affect this
translation. Thus, we expect that a drive reducing mean fit-
ness to 0.5 will have largely the same population impact re-
gardless of whether the drive is a 1- or 2-sex homing drive or
a toxin-antidote drive (assuming no resistance). Fitness suppression potential across different drive systems
423 Thus,
442
as a consequence of c/C polymorphism at equilibrium (noted
443
above), the most extreme fitness suppression occurs when both
444
homozygotes die, leaving Cc heterozygotes as the only viable
445
genotype and a mean fitness of 0.5 (Fig. 4). 446 Figure 3 Equilibrium mean fitness (average survival) for toxin-
antidote (ClvR) evolution when confronted with evolution of
sib mating. Panels differ in the level m of sib mating imposed
by the inbreeding allele: 0.2 (top panel), 0.5 (middle panel),
0.95 (bottom panel). The blue curve represents equilibrium
mean fitness when ClvR evolves without interference from sib
mating, 1/(1 + σ). Sib mating evolves to suppress the drive
where the black curve rises above the blue; the black curve
represents evolved mean fitness. In contrast to sib mating sup-
pression of homing drives, the effect on ClvR is close to all or
none – mean fitness is virtually always 1.0 or that expected
if ClvR is unaffected. The shaded area spans the values of s
for which type-M resistance evolves in the same model (even
though s and σ have somewhat different effects). As with hom-
ing drives, sib mating can evolve at much smaller values of
σ if the sib mating allele enforces a high level of sib mating. Initial frequencies of the ClvR and sib-mating alleles were 0.07;
initial frequencies in the type-M trials were 0.01. Trials were
evaluated at 20,000 generations. The figure shows that 2-sex homing drives can achieve far
4
greater fitness suppression than either 1-sex homing drives or
4
ClvR. Perhaps surprisingly, there is little difference between 1-
4
sex and 2-sex drives much less difference in the resistance-free
4
zone of fitness suppression (Gomulkiewicz et al. 2021). In the
4
preceding sections of this paper, our sib-mating models were
4
effectively 2-sex drives, but the section comparing two drives
4
with one drive assumed a 1-sex drive. The conclusions of those
4
sections should not be qualitatively sensitive to the assumptions
4
of 1-sex versus 2-sex drives. 4 The next section delves further into ecology but continues
457
with the theme of resistance evolution. It is limited to homing
458
drives, but the principles should apply to toxin-antidote drives
459
as well. 460 Cook et al. Avoiding resistance using ecology: homing drives ;
https://doi.org/10.1101/2021.08.30.458221
doi:
bioRxiv preprint ramifications of this latter point are clear: evolution of resistance
532
against a drive with a density-dependent fitness effect can be
533
avoided by maintaining the population in a suppressed state
534
that largely avoids the density-dependent effects. 535 well as a density-dependent manner (Table 6). The density-
499
dependent effects are analogous to effects on ecological carrying
500
capacity (though not strictly the same). Following previous
501
sections of this paper, our basic question is how gene drive
502
avoidance of resistance depends on an ecological context. Can
503
the ecology be used to further evade resistance? 504 relative
relative
population size
density-ind. density-dep. (progeny)
viability of AA
viability of AA
N < K(1 −sK)
1 −sb
1
K(1 −sK) ≤N ≤K
1 −sb
K(1−sK)
N
K < N
1 −sb
1 −sK drive
mother’s
density-ind. density-dep. genotype
birth rate
viability
viability
a−
b
1
min (1, K
N )
AA
b
1 −sb
min (1, K(1−sK)
N
) Table 6 Genotype-specific birth rate and progeny viability
in the homing drive model of ecological effects. A mother’s
number of surviving offspring is the product of her births (b,
genotype-independent) and the survival of her offspring ac-
cording to their genotypes. The net survival of each progeny is
the product of a density-independent and density-dependent
term (given in the third and fourth columns) and depends
on their genotypes. The density-dependent model is a type
known as ‘ceiling’ density dependence (Lande 1993). N is the
total number of progeny born in the population that gener-
ation, prior to viability effects. K is the threshold density of
N for the influence of density-dependent effects. A birth rate
term is included here to emphasize that the birth rate affects
the population dynamics and evolution even though there is
no genotypic effect on birth rate. These rules used for enforc-
ing density dependence are convenient for numerical analysis,
and they have the advantage of preventing major fluctuations
in adult population sizes that can occur with many other types
of density dependence. Table 7 Relative fitnesses of AA genotypes as determined by
offspring viability and according to the number of progeny
born in the population in that generation (N); these relative
viabilities follow directly from Table 6 but are presented here
for clarity. Avoiding resistance using ecology: homing drives There are 3 zones depending on the relative mag-
nitudes of N, K, and K(1 −sK), and the zones apply to the
density-dependent component. When the number of offspring
born is below K(1 −sK), all genotypes (aa, Aa, and AA) have
equal density-dependent fitness components; the relative fit-
ness of AA declines as the number of offspring increases, but
only down to 1 −sK – when the absolute viability of AA is
K(1 −sK)/N but the absolute viabilities of aa and Aa are K/N. In contrast, the density-independent viability component is
constant across all three zones. These results are so far limited to the evolution of resistance
536
and then only showed final outcomes. The parameters were
537
chosen so that none of the trials led to population extinction,
538
but the evolution did sometimes result in suppressed densities. 539
We now show how extinction can occur, finding that extinction
540
can be achieved by a density-independent effect even when the
541
density-dependent effect would allow persistence – all while
542
avoiding resistance evolution. 543 Much of the evolution in this model can be qualitatively an-
ticipated by translating the information of Table 6 into density-
dependent effects on just the drive homozygote, AA. Its density-
dependent viability descends from 1 to 1 −sK across three zones
determined by the population size N relative to the thresholds
K and K(1 −sK) (Table 7). Comparing density-dependent to
density-independent viability (for sb = sK), density-dependent
selection against AA is never more extreme than density-
independent selection and may be considerably less. Whenever
N is below the AA viability threshold of K(1 −sK), the drive has
no relative fitness effect on AA – providing a ready means for
ensuring resistance-free evolution by (for example) suppressing
population size artificially. From the above results, it is easy to appreciate that drive-
544
caused extinction from a density-independent viability effect
545
requires an intrinsically low birth rate. To avoid resistance, the
546
drive cannot have too large of a viability reduction, sb (an ap-
547
proximate upper limit is 0.4 for these models, Fig. 5). Therefore,
548
when the intrinsic birth rate b is near the minimum for popu-
549
lation persistence, a density-independent sb = 0.4 can evolve
550
resistance-free and suppress viability below that needed to main-
551
tain the population (Fig. Avoiding resistance using ecology: homing drives There will be
differences in the speed of evolution, and speed could affect the
evolutionary response and/or the ecology. Figure 4 Equilibrium mean fitness (average survival) as a func-
tion of the drive homozygote fitness effect for single 1-sex
homing drives (purple), 2-sex homing drives (green), and the
ClvR toxin-antidote drive (blue); s applies to homing drives,
σ to ClvR. These outcomes assume that the fitness effect oper-
ates in both sexes, that drive has evolved to its maximum level,
and that resistance is absent; similar curves apply if the fitness
effect is confined to one sex and mean fitness is measured in
that sex. Only 2-sex homing drives can suppress mean fitness
below 0.5 for these systems, although some toxin-antidote sys-
tems can suppress mean fitness even lower (Champer et al. 2020a). A comparison of fitness effects across different drive
systems is also provided by Beaghton et al. (2019). There are countless possibilities to consider at the interface
of gene drives and ecology. Our focus continues to be that of
avoiding resistance evolution. We develop a model to address
the relationship between population genetics of gene drives, re-
sistance evolution, and ecological impact on population. The
model is deterministic and assumes discrete generations, ran-
dom mating of diploid males and females, with drive operating
only in (heterozygous) males. To model population size, the
number of progeny born is determined by the number of adult
females; males are never limiting for female reproduction. Birth
rates and viability effects on females therefore determine popu-
lation size in each generation based on numbers of adult females
in the previous generation. Fitness effects of the drive affect progeny viability regardless
of sex and may operate in a density-independent manner as Cook et al. 8 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 4, 2022. Avoiding resistance using ecology: homing drives The DI bars combine
many trials using different birth rates, as all resulted in the
same resistance-free upper limit. (Top) Population numbers
were determined solely by births and homing drive evolution,
without any outside suppression or enhancement of num-
bers. All drives, whether affecting the density-independent
or density-dependent viability parameter, showed the same
resistance-free upper limit on evolution (approximately 0.43). (Bottom) Trials used the same conditions as in the top panel,
except that total progeny numbers were artificially depressed
every 5 generations to 1/4 of their initial values. Depending
on the intrinsic birth rate, this depression could lower the total
progeny (N) below the thresholds K and K(1 −sK) in Table 6. Furthermore, again depending on birth rates, the population
could require several generations to recover before the next
suppression. This suppression had no effect on the resistance-
free upper limit when density-independent viability operated. However, with density-dependent viability effects, the range
of resistance-free sK increased as b declined. This latter effect
of b is expected because the temporary depression of popula-
tion numbers is greater and the recovery slower with lower
birth rates, hence the population experiences longer periods
of reduced selection against the drive allele (as per Table 7)
which in turn reduces selection for resistance (Gomulkiewicz
et al. 2021). (Caption continued on next column.) 0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
Population Dynamics
generation
relative density
Density dependent
Density independent
Wild-type K Population Dynamics Figure 6 Drives with density-independent fitness effects can
cause extinction when drives with the equivalent density-
dependent fitness effects do not. Extinction requires a low
intrinsic birth rate, so that the drive can evade resistance by
having only a moderate fitness effect yet suppress births below
that needed for population persistence. In contrast to other
figures in this paper, the population dynamics over time are
illustrated. The top panel shows the evolutionary dynam-
ics of drive allele frequency (green) and of resistance allele
frequency (blue) for the density-independent (dashed) and
density-dependent (solid) cases. The evolution is approxi-
mately the same in both models. The bottom panel displays
the dynamics of population densities (relative to K) for the two
cases. Extinction occurs for the density-independent case as
the density approaches zero, but not in the density-dependent
case, for which the density declines by a factor 1 −sK. Avoiding resistance using ecology: homing drives This latter effect
of b is expected because the temporary depression of popula-
tion numbers is greater and the recovery slower with lower
birth rates, hence the population experiences longer periods
of reduced selection against the drive allele (as per Table 7)
which in turn reduces selection for resistance (Gomulkiewicz
Figure 5 (continued) Parameter values and initial conditions
were K = 1 × 109, initial population size 1 × 109, and ini-
tial frequencies of drive and resistance allele = 0.0005. The
slight difference between the resistance-free zone of density-
independent effects here and in Fig. 1 is due to the different
initial frequencies used. 0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
Evolution
generation
frequency
Density dependent
Density independent
Drive allele
Resistance allele
0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
Population Dynamics
generation
relative density
Density dependent
Density independent
Wild-type K
Figure 6 Drives with density-independent fitness effects can
cause extinction when drives with the equivalent density-
dependent fitness effects do not. Extinction requires a low
intrinsic birth rate, so that the drive can evade resistance by
having only a moderate fitness effect yet suppress births below
that needed for population persistence. In contrast to other
figures in this paper, the population dynamics over time are
illustrated. The top panel shows the evolutionary dynam-
ics of drive allele frequency (green) and of resistance allele
frequency (blue) for the density-independent (dashed) and
density-dependent (solid) cases. The evolution is approxi-
mately the same in both models. The bottom panel displays
the dynamics of population densities (relative to K) for the two
cases. Extinction occurs for the density-independent case as
the density approaches zero, but not in the density-dependent
case for which the density declines by a factor 1
sK Param Figure 5 (continued) Parameter values and initial conditions
were K = 1 × 109, initial population size 1 × 109, and ini-
tial frequencies of drive and resistance allele = 0.0005. The
slight difference between the resistance-free zone of density-
independent effects here and in Fig. 1 is due to the different
initial frequencies used. Avoiding resistance using ecology: homing drives Resistance-proof sb or sk
0 0
0 2
0 4
0 6
0 8
1 0 0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
Evolution
generation
frequency
Density dependent
Density independent
Drive allele
Resistance allele
0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
Population Dynamics
generation
relative density
Density dependent
Density independent
Wild-type K
Figure 6 Drives with density-independent fitness effects can
cause extinction when drives with the equivalent density-
dependent fitness effects do not. Extinction requires a low
intrinsic birth rate, so that the drive can evade resistance by
having only a moderate fitness effect yet suppress births below
that needed for population persistence. In contrast to other
figures in this paper, the population dynamics over time are
illustrated. The top panel shows the evolutionary dynam-
ics of drive allele frequency (green) and of resistance allele
frequency (blue) for the density-independent (dashed) and
density-dependent (solid) cases. The evolution is approxi-
mately the same in both models. The bottom panel displays
the dynamics of population densities (relative to K) for the two
cases. Extinction occurs for the density-independent case as
the density approaches zero, but not in the density-dependent
case, for which the density declines by a factor 1 −sK. Param-
eter values are b = 3, sb = sK = 0.4, K = 1 × 109. Initial allele
frequencies were the same as in Fig. 5. 0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
Evolution
generation
frequency
Density dependent
Density independent
Drive allele
Resistance allele Evolution Evolution DI b>3
DD b = 15
DD b = 10
DD b = 4
Model with knockdown
Resistance-proof sb or sk
0.0
0.2
0.4
0.6
0.8
1.0 Figure 5 A drive that suppresses viability can evade resistance
at higher fitness effects when its effect is density-dependent
than when its effect is density-independent. Bars show the
range of viability coefficients (sb, sK, Table 6) that allowed
resistance-free evolution of a homing drive in the ecology
model. The bars on the left of each panel (red, labeled with
DI) apply to all trials in which the drive affected density-
independent viability (sb); the three (green) bars on the
right apply to trials in which the drive affected the density-
dependent viability (sK), each specific to a different value of
the intrinsic birth rate (b), as indicated. Avoiding resistance using ecology: homing drives 6); in contrast, if the intrinsic birth rate
552
is high, this evolution will not lead to extinction. Perhaps surpris-
553
ingly, under the very conditions that will cause extinction when
554
the viability effect is density-independent, assigning the same
555
viability effect to sK instead of sb will lead to lower adult popu-
556
lation size, not extinction (Fig. 6). We suggest that this inability
557
to cause extinction through density-dependent viability will be
558
general in this (deterministic) model for any reasonable value of
559
K. However, adding Allee effects or demographic stochasticity
560
could lead to extinction with suppression of density-dependent
561
viability (Lande et al. 2003). 562 p p
y
To study the impact of both types of viability effects on
resistance-free evolution, numerical trials were conducted with
different birth rates and with periodic depression of the popula-
tion’s progeny; further model details are given in figure legends
and tables. The clear result is that a homing drive with purely
density-independent effects evades resistance according to the
usual rules from population genetics models (Fig. 5, top). In
contrast, a drive with purely density-dependent effects evolves
differently; it evades resistance more easily when the population
is frequently suppressed below its maximum. With the periodic
population culling (knockdown) imposed by our model, resis-
tance evasion is further enhanced by low birth rates, because low
birth delays the population’s rebound to the viability threshold
K (Fig. 5, bottom). As elaborated in the Discussion, the practical The ecology models developed here allow us to tentatively
563
identify principles for resistance avoidance:
564 • Density-dependent fitness effects afford opportunities to
565 Resistance proofing gene drives 9 . CC-BY 4.0 International license
available under a
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doi:
bioRxiv preprint DI b>3
DD b = 15
DD b = 10
DD b = 4
Model without knockdown
Resistance-proof sb or sk
0.0
0.2
0.4
0.6
0.8
1.0
DI b>3
DD b = 15
DD b = 10
DD b = 4
Model with knockdown
Resistance-proof sb or sk
0.0
0.2
0.4
0.6
0.8
1.0
Figure 5 A drive that suppresses viability can evade resistance
at higher fitness effects when its effect is density-dependent
than when its effect is density-independent. Bars show the
range of viability coefficients (sb, sK, Table 6) that allowed
resistance-free evolution of a homing drive in the ecology
model. The bars on the left of each panel (red, labeled with
DI) apply to all trials in which the drive affected density-
independent viability (sb); the three (green) bars on the
right apply to trials in which the drive affected the density-
dependent viability (sK), each specific to a different value of
the intrinsic birth rate (b), as indicated. The DI bars combine
many trials using different birth rates, as all resulted in the
same resistance-free upper limit. (Top) Population numbers
were determined solely by births and homing drive evolution,
without any outside suppression or enhancement of num-
bers. All drives, whether affecting the density-independent
or density-dependent viability parameter, showed the same
resistance-free upper limit on evolution (approximately 0.43). (Bottom) Trials used the same conditions as in the top panel,
except that total progeny numbers were artificially depressed
every 5 generations to 1/4 of their initial values. Depending
on the intrinsic birth rate, this depression could lower the total
progeny (N) below the thresholds K and K(1 −sK) in Table 6. Furthermore, again depending on birth rates, the population
could require several generations to recover before the next
suppression. This suppression had no effect on the resistance-
free upper limit when density-independent viability operated. However, with density-dependent viability effects, the range
of resistance-free sK increased as b declined. Cook et al. Discussion
591 At face value, the easy engineering of gene drives affords an
592
ability to manipulate plant and animal pests that is unprece-
593
dented in the history of civilization. Merely releasing a handful
594
of appropriately-engineered individuals of a species can poten-
595
tially eradicate the entire species worldwide using suppression
596
drives. This potential was known at least for the last half cen-
597
tury (Hamilton 1967; Lyttle 1977), but engineering remained the
598
hurdle. With engineering no longer a hurdle in gene drive con-
599
struction, (e.g., Kyrou et al. 2018; Oberhofer et al. 2019; Champer
600
et al. 2020a,c; Oberhofer et al. 2020; Simoni et al. 2020; Bier 2021),
601
the unresolved question is whether resistance evolution will
602
thwart the effort, as observed in some cage populations of the
603
first experimental gene drive study (Lyttle 1979, 1981), but not
604
invariably in more recent ones (Kyrou et al. 2018; Hammond
605
et al. 2017). It may be impossible to accurately predict resistance
606
evolution in any wild release (Burt 2003), since the outcome will
607
depend on the nature of variation in the species, but modeling
608
can at least help us to anticipate possibilities. 609 Despite the limited number of studies available, a distilled
663
interpretation of them is that evasion of resistance evolution
664
by a suppression drive is feasible but not assured. The most
665
important consideration is to avoid ‘allelic’ resistance, which for
666
a homing drive would typically be a mutation in the nuclease
667
cleavage site. There are now two feasible ways around that, one
668
being to choose a cleavage site whose function is intolerant of
669
mutation (Kyrou et al. 2018), the other being to take advantage
670
of CRISPR to target multiple, independent sites in the same
671
gene such that every chromosome in the population will have
672
at least one site susceptible (Noble et al. 2017; Champer et al. 673
2018, 2020d; Edgington et al. 2020). The latter approach would
674
greatly expand the opportunities for population suppression,
675
but whether either approach will succeed may depend on suc-
676
cessfully controlling expression of CRISPR proteins to avoid
677
undesirable embryonic and somatic activity (e.g., Champer et al. 678
2017; Bier 2021; Hammond et al. 2021)). Avoiding resistance using ecology: homing drives Param-
eter values are b = 3, sb = sK = 0.4, K = 1 × 109. Initial allele
frequencies were the same as in Fig. 5. Cook et al. 10 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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bioRxiv preprint . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 4, 2022. ;
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doi:
bioRxiv preprint tion depended on linkage disequilibrium between the resistance
627
allele and drive-sensitive allele, and for moderate-effect drives
628
and sufficiently low initial frequencies of the resistance allele,
629
the drive allele essentially outran resistance to fixation. Once the
630
drive fixed, type-M resistance had no effect. 631 relax resistance evolution. For example, drive-imposed
lethality that operates only at high population density ex-
periences reduced resistance evolution if the population
is temporarily maintained at low density until the drive
has swept. This population suppression can be manifest
artificially or (in some cases) by the gene drive itself, the
latter process being sensitive to intrinsic birth rates. Like-
wise, conditionally expressed fitness effects can avoid se-
lection during drive spread and thus facilitate avoidance
of resistance despite later manifestation of the fitness ef-
fects. For example, a drive that reverses pesticide resistance
can evolve cost-free if pesticide use is averted until drive
fixation. yp
The work here expands on the potential robustness of
632
resistance-free evolution of unlinked suppression drives. Three
633
meaningful elaborations can be offered:
634 • With homing drives, population suppression while avoid-
635
ing resistance of type M can be increased by using multiple
636
drives with individually mild effects instead of a single
637
drive with the same total effect. 638 nce of type M can be increased by using multiple • Sib mating (type-P resistance) can evolve to suppress
639
moderate-effect drives, but only if genotypes exist that en-
640
force high levels of sib mating. Avoiding resistance using ecology: homing drives High levels of sib mating
641
can evolve despite being rare in the population, but low
642
genetic levels of sib mating are not favored except with
643
strong-effect drives. These results apply to both homing
644
drives and toxin-antidote drives (here represented by ClvR). 645 • Density-independent fitness effects can achieve extinction
while avoiding resistance evolution if intrinsic fecundity
is low. There may be few pest species for which this is
relevant though it may help with suppression of naturalized
invasives. • Fitness effects that operate in only one sex reduce resistance
evolution while possibly enhancing or minimizing ecolog-
ical effects. Thus, reducing only female fitness will more
easily suppress populations than does reducing only male
fitness, and fitness effects confined to one sex reduces se-
lection for resistance (e.g., Fig. 1, and (Gomulkiewicz et al. 2021)). • The realm of resistance-free evolution can be expanded by
646
manipulating the ecological effects of the drive. Drives
647
with density-dependent fitness effects can escape much of
648
resistance evolution by externally imposed suppression of
649
the population or by taking advantage of natural declines
650
in population size (e.g., seasonality). 651 There are yet many elaborations to explore with the mod-
652
els, especially on the ecological end. Sib mating might increase
653
dynamically (not through evolution) as the population size de-
654
clines. Group selection itself (or its equivalent in a continuous
655
population across space) may slow or even stop the spread of
656
the drive (Bull et al. 2019; Champer et al. 2021). Our efforts
657
here considered toxin-antidote drives (ClvR) in only a limited
658
scope, and although there was nothing in our results to sug-
659
gest a fundamental difference between resistance evolution for
660
homing drives than for toxin-antidote drives, a more thorough
661
consideration of toxin-antidote drives is warranted. 662 Resistance proofing gene drives Discussion
591 CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 4, 2022. ;
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doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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bioRxiv preprint block moderate-effect drives unless deliberately engineered to
689
be closely linked to the drive or if introduced at high frequency. 690 armadillo/Gene_drives_C_Files_papers. The models calculate
749
offspring genotype numbers by exhaustive enumeration of all
750
possible parental matings multiplied by the fraction of gamete
751
or offspring types produced by the parental genotypes; there are
752
no explicit recursions of genotype frequencies in one generation
753
from their frequencies in previous generations. 754 y
g
q
y
Although suppression drives of moderate-effect may be the
best bet for avoiding resistance evolution, they present some em-
pirical difficulties. It is obvious that a suppression drive is only
likely to succeed if it operates by destroying target gene function
instead of carrying a deleterious cargo. Loss of a deleterious
cargo would be a simple and evolutionarily unavoidable way to
eliminate the suppressive effect of the drive. Furthermore, any
genomic site chosen for disruption by a moderate-effect drive
must be present in all members of the species, but it cannot be
a strictly essential gene, for which null homozygotes would be
inviable or sterile. This may raise the challenge of knowing the
fitness effects of a null homozygote in advance of the release. Being assured of a fitness effect of 0.4 (for example) across the
heterogeneous environment in the wild is not practical. Conclusion
722 A wide variety of gene drives can now be engineered, and ex-
periments with model organisms in laboratory settings confirm
their potential utility in suppressing populations. The unknown
that faces releases into natural populations is the evolution of
resistance. We previously showed that suppression drives with
moderate fitness effects can evade many types of resistance that
are unlinked to the drive locus. Here we have identified addi-
tional ways of escaping unlinked resistance. One method is to
introduce multiple (e.g., two) drives of individually mild effect
to achieve a greater combined suppression. Another method
is to rely on the fact that resistance evolves in response to fit-
ness effects of the drive; careful choice of ecological effects of the
drive may thus enable population suppression with only modest
fitness effects. One remaining challenge is that sib mating can
evolve in response to a suppression drive with any fitness effect;
sib mating can limit the suppression and even cause loss of the
drive. However, sib mating is a threat for moderate-effect sup-
pression drives only from alleles for high levels of sib mating (or
if sib mating arises as a demographic effect of the reduction in
population size). Evolution of resistance to suppression drives
may therefore cause problems only on a limited basis. Burt, A., 2003 Site-specific selfish genes as tools for the control
782
and genetic engineering of natural populations. Proceedings. 783
Biological Sciences / The Royal Society 270: 921–928. 784 g
y
y
Champer, J., I. K. Kim, S. E. Champer, A. G. Clark, and P. W. 785
Messer, 2020a Performance analysis of novel toxin-antidote
786
CRISPR gene drive systems. BMC Biology 18: 27. 787 Champer, J., I. K. Kim, S. E. Champer, A. G. Clark, and P. W. 788
Messer, 2021 Suppression gene drive in continuous space can
789
result in unstable persistence of both drive and wild-type
790
alleles. Molecular Ecology 30: 1086–1101. 791 Champer, J., E. Lee, E. Yang, C. Liu, A. G. Clark, et al., 2020b A
792
toxin-antidote CRISPR gene drive system for regional popula-
793
tion modification. Nature Communications 11: 1082, Number:
794
1 Publisher: Nature Publishing Group. 795 g
p
Champer, J., J. Liu, S. Y. Oh, R. Reeves, A. Luthra, et al., 2018 Re-
796
ducing resistance allele formation in CRISPR gene drive. Pro-
797
ceedings of the National Academy of Sciences of the United
798
States of America 115: 5522–5527. Acknowledgments We thank Jackson Champer for discussion and references. An
756
anonymous reviewer prompted us to consider the effects of
757
imperfect drive and heterozygote fitness effects and provided
758
references. RG was supported by a WSU Honors College Distin-
759
guished Professorship, FC by a University of Idaho funds, and
760
JJB by NIH grant GM 122079. 761 Discussion
591 Then,
when the population persists after fixation of a moderate-effect
drive (as will usually be the case), compensatory evolution can
be expected to reduce the fitness effect of the knockout – type-
C resistance Hall (2003); Rokyta et al. (2002); Harcombe et al. (2009); van Leeuwen et al. (2020). Literature Cited Beaghton, A., P. J. Beaghton, and A. Burt, 2017 Vector control
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with driving Y chromosomes: modelling the evolution of
764
resistance. Malaria Journal 16: 286. 765 Beaghton, A. K., A. Hammond, T. Nolan, A. Crisanti, and A. Burt,
766
2019 Gene drive for population genetic control: non-functional
767
resistance and parental effects. Proceedings. Biological Sci-
768
ences 286: 20191586. 769 Official releases of synthetic gene drives have yet to be ap-
proved. Any release of a suppression drive should anticipate
the potential for resistance evolution at least because some types
of resistance, once evolved, will thwart other attempts with sup-
pression drives (e.g., type P). Thus the failure of one suppression
drive may ensure the failure of subsequent drives. However,
delaying the approval of official releases has its own risk in an
age when multiple labs can create gene drives – the uninten-
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591 Other forms of type-M
679
resistance, such as interference with nuclease expression and
680
function, are not likely to be allelic or even closely linked to the
681
drive, but chromosomal rearrangements could change linkage
682
– to lock distant loci into a single linkage group. And if un-
683
linked type-M and type-P resistance doesn’t already exist in the
684
population, they are not likely to arise in time to stop a strong
685
drive. However, deliberate introduction and expression of an
686
anti-CRISPR protein, which may offer a fail-safe block against
687
extreme suppression drives (Taxiarchi et al. 2021), may fail to
688 p
p
p
The conclusion here and in a previous study (Gomulkiewicz
610
et al. 2021) is that engineering can substantially influence
611
whether resistance evolves to block a suppression drive. Use of
612
a highly suppressive drive will ensure evolution of resistance if
613
resistance exists in the population or can arise quickly enough. 614
Use of a moderately suppressive drive can escape resistance
615
evolution. This paper elaborates on some of this understanding. 616
We distinguished three types of resistance evolution: resis-
617
tance that blocks the drive mechanism (type M, Lyttle 1979;
618
Champer et al. 2017), compensatory resistance that reverses the
619
fitness effects of the drive without affecting drive (type C, as
620
observed by (Lyttle 1981)), and resistance that affects population
621
structure to prevent the drive from spreading (type P). We pre-
622
viously showed that suppression drives with moderate fitness
623
effects could escape unlinked type-M resistance evolution even
624
when the resistance alleles were present initially and imposed
625
no cost on fitness (Gomulkiewicz et al. 2021). Resistance evolu-
626 We distinguished three types of resistance evolution: resis-
617
tance that blocks the drive mechanism (type M, Lyttle 1979;
618
Champer et al. 2017), compensatory resistance that reverses the
619
fitness effects of the drive without affecting drive (type C, as
620
observed by (Lyttle 1981)), and resistance that affects population
621
structure to prevent the drive from spreading (type P). We pre-
622
viously showed that suppression drives with moderate fitness
623
effects could escape unlinked type-M resistance evolution even
624
when the resistance alleles were present initially and imposed
625
no cost on fitness (Gomulkiewicz et al. 2021). Resistance evolu-
626 11 . Cook et al. Conclusion
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 4, 2022.
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https://doi.org/10.1101/2021.08.30.458221
doi:
bioRxiv preprint .
CC-BY 4.0 International license
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this version posted February 4, 2022.
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869 Resistance proofing gene drives 13 . CC-BY 4.0 International license
available under a
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Small Bowel Perforation as a Postoperative Complication from a Laminectomy
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Case reports in surgery
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Hindawi Publishing Corporation
Case Reports in Surgery
Volume 2015, Article ID 378218, 4 pages
http://dx.doi.org/10.1155/2015/378218 Hindawi Publishing Corporation
Case Reports in Surgery
Volume 2015, Article ID 378218, 4 pages
http://dx.doi.org/10.1155/2015/378218 Hindawi Publishing Corporation
Case Reports in Surgery
Volume 2015, Article ID 378218, 4 pages
http://dx.doi.org/10.1155/2015/378218 Robert H. Krieger,1 Katherine M. Wojcicki,1 Andrew C. Berry,2
Warren L. Reuther III,3 and Kendrick D. McArthur4 Robert H. Krieger,1 Katherine M. Wojcicki,1 Andrew C. Berry,2
Warren L. Reuther III,3 and Kendrick D. McArthur4
1Kansas City University of Medicine and Biosciences (KCU), 1750 E. Independence Avenue, Kansas City, MO 64106, USA
2Department of Internal Medicine, University of South Alabama, Mobile, AL 36608, USA
3Department of Radiology, West Palm Hospital, West Palm Beach, FL 33407, USA
4Department of Surgery, West Palm Hospital, West Palm Beach, FL 33407, USA 1Kansas City University of Medicine and Biosciences (KCU), 1750 E. Independence Avenue, Kansas City, MO 64106, USA
2Department of Internal Medicine, University of South Alabama, Mobile, AL 36608, USA
3Department of Radiology, West Palm Hospital, West Palm Beach, FL 33407, USA
4Department of Surgery, West Palm Hospital, West Palm Beach, FL 33407, USA 1Kansas City University of Medicine and Biosciences (KCU), 1750 E. Independence Avenue, Kansas City, MO 64106, USA
2Department of Internal Medicine, University of South Alabama, Mobile, AL 36608, USA
3Department of Radiology, West Palm Hospital, West Palm Beach, FL 33407, USA
4Department of Surgery, West Palm Hospital, West Palm Beach, FL 33407, USA Correspondence should be addressed to Robert H. Krieger; rkrieger@kcumb.edu Received 19 March 2015; Accepted 28 June 2015 Received 19 March 2015; Accepted 28 June 2015 Academic Editor: Yoshiharu Kawaguchi Copyright © 2015 Robert H. Krieger et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. Chronic low back pain is one of the leading chief complaints affecting adults in the United States. As a result, this increases the
percentage of patients that will eventually undergo surgical intervention to alleviate debilitating, chronic symptoms. A 37-year-old
woman presented ten hours postoperatively after a lumbar laminectomy with an acute abdomen due to the extraordinarily rare
complication of small bowel injury secondary to deep surgical penetration. 1. Introduction a review of the literature revealed that this uncommon
complication is more likely during a discectomy when com-
pared to a laminectomy, which makes our reported case an
exceptionally rare report [7]. In the United States, back pain remains the second most com-
mon reason why patients visit a clinician and up to 84 percent
of adults will eventually endure low back pain [1, 2]. Approx-
imately 250,000–500,000 people in the United States have
symptoms of spinal stenosis, and although many patients
remain asymptomatic to mildly symptomatic, the prevalence
of symptomatic back pain is significant enough to warrant
neurosurgical intervention and case volume will remain
steady [3]. Cohort studies of individuals with lumbar spinal
stenosis (LSS) demonstrated that 30 percent of the patients
subsequently requested surgical treatment after initially
choosing nonsurgical, medical management [4, 5]. As one
would expect, surgery to the vertebral column is not without
risk and although ventral perforation is rare, injury to the
retroperitoneal vessels is the most common, serious, com-
plication in this scenario [6]. However, this case illustrates
one of the rare instances where the small bowel was injured
during a lumbar laminectomy and highlights the importance
of recognizing the acute abdomen as a potentially lethal
complication of a laminectomy or discectomy. Furthermore, Case Report
Small Bowel Perforation as a Postoperative
Complication from a Laminectomy Robert H. Krieger,1 Katherine M. Wojcicki,1 Andrew C. Berry,2
Warren L. Reuther III,3 and Kendrick D. McArthur4
1Kansas City University of Medicine and Biosciences (KCU), 1750 E. Independence Avenue, Kansas City, MO 64106, USA
2Department of Internal Medicine, University of South Alabama, Mobile, AL 36608, USA
3Department of Radiology, West Palm Hospital, West Palm Beach, FL 33407, USA
4Department of Surgery, West Palm Hospital, West Palm Beach, FL 33407, USA 2. Case Report (b) The arrow pointing to the site of perforation located at the approximate jejunal-ileal junction. (a) (b) (a) Figure 2: Laparoscopic views of the inflamed and perforated small bowel photographed prior to the conversion to laparotomy. (a) The
photograph demonstrates a highly erythematous, inflamed segment of small bowel with fibrinous exudation and inflammation adjacent to
the instrument. (b) The arrow pointing to the site of perforation located at the approximate jejunal-ileal junction. that her abdomen was exquisitely tender to palpation without
any distension and there was significant tympany upon
percussion. The electrolyte profile was essentially normal, but
total bilirubin was elevated at 2.1 mg/dL. Urine pregnancy
test was negative and the complete blood count showed an
elevated white blood cell count of 15.4 × 103/𝜇L, with a
hemoglobin and hematocrit of 12 g/dL and 34%, respectively. Computed tomography (CT) of the abdomen and pelvis
showed evidence of retroperitoneal air, which seemingly
tracked back into the spinal canal (Figure 1(a)). In addition,
there was free air seen intraperitoneally without an obvi-
ous source or evidence of an inflammatory process in her
abdomen at that time (Figure 1(b)). It was suspected that
the retroperitoneal air may be secondary to deep surgical
penetration into the small bowel during the laminectomy and
the patient was initially treated expectantly with intravenous
antibiotics and observation. On hospital course days two and
three the patient began to spike and maintain fevers, with air that her abdomen was exquisitely tender to palpation without
any distension and there was significant tympany upon
percussion. The electrolyte profile was essentially normal, but
total bilirubin was elevated at 2.1 mg/dL. Urine pregnancy
test was negative and the complete blood count showed an
elevated white blood cell count of 15.4 × 103/𝜇L, with a
hemoglobin and hematocrit of 12 g/dL and 34%, respectively. continuing to appear in the psoas muscles and significant air
within the peritoneal cavity. The follow-up CT showed that
the patient had developed ascites suggesting a small bowel
perforation until proven otherwise (Figure 1(c)). The patient
was taken to the operating room for emergent laparoscopic
exploration. After dissecting through the subcutaneous tissue and the
anterior rectus fascia in a standard fashion, the Hassan trocar
was inserted and a laparoscopic view of the abdomen was
undertaken. There was a marked amount of seropurulent
fluid extending from the right and left colic gutters down
to the Pouch of Douglas. 2. Case Report A 37-year-old female, postlaminectomy status for the treat-
ment of lumbar back pain, presented with unrelenting
abdominal pain 10 hours after the procedure. The patient
denied any nausea, vomiting, diarrhea, fevers, or chills at that
time, but due to the acute nature of the pain it was evident
to her that she had either developed some type of postoper-
ative complication or was experiencing a nascent abdominal
pathology that would require immediate medical attention. The patient’s past medical history was significant for chronic
back pain and lumbar disc disease, with no significant surgi-
cal history prior to the laminectomy. Vital signs upon admis-
sion and consultation were stable and the patient was afebrile. Physical exam revealed a well-nourished, well-developed
female that was alert and oriented albeit in obvious discom-
fort. The patient’s physical exam was unremarkable except 2 Case Reports in Surgery (a)
(b)
(c)
Figure 1: Computed tomography (CT) of the abdomen/pelvis demonstrates the progression of intra-abdominal injury and free air
accumulation. (a) Transaxial view highlighting the accumulation of air in the spinal canal, psoas muscles, and the retroperitoneum, secondary
to deep penetration and injury of the small bowel during the laminectomy. (b) This axial view demonstrates three distinct locations (labeled
1, 2, and 3) of intra-abdominal free air found on a follow-up CT of the abdomen/pelvis. (c) Axial view depicting extraluminal air anterior to
the bowel, as well as air within the mesentery and posterior to the right psoas muscle. (b) (c) (a) (a) (b) (c) Figure 1: Computed tomography (CT) of the abdomen/pelvis demonstrates the progression of intra-abdominal injury and free air
accumulation. (a) Transaxial view highlighting the accumulation of air in the spinal canal, psoas muscles, and the retroperitoneum, secondary
to deep penetration and injury of the small bowel during the laminectomy. (b) This axial view demonstrates three distinct locations (labeled
1, 2, and 3) of intra-abdominal free air found on a follow-up CT of the abdomen/pelvis. (c) Axial view depicting extraluminal air anterior to
the bowel, as well as air within the mesentery and posterior to the right psoas muscle. (a)
(b)
Figure 2: Laparoscopic views of the inflamed and perforated small bowel photographed prior to the conversion to laparotomy. (a) The
photograph demonstrates a highly erythematous, inflamed segment of small bowel with fibrinous exudation and inflammation adjacent to
the instrument. 2. Case Report The appendix was visualized
and appeared normal; however, while running the small
bowel, beginning at the terminal ileum, a significant amount
of inflammatory and fibrinous exudate was discovered
(Figure 2(a)). Upon approaching the proximal ileum and
jejunum, an area of perforation was identified as evidenced
by where the omentum had adhered to the small bowel Computed tomography (CT) of the abdomen and pelvis
showed evidence of retroperitoneal air, which seemingly
tracked back into the spinal canal (Figure 1(a)). In addition,
there was free air seen intraperitoneally without an obvi-
ous source or evidence of an inflammatory process in her
abdomen at that time (Figure 1(b)). It was suspected that
the retroperitoneal air may be secondary to deep surgical
penetration into the small bowel during the laminectomy and
the patient was initially treated expectantly with intravenous
antibiotics and observation. On hospital course days two and
three the patient began to spike and maintain fevers, with air 3 Case Reports in Surgery A delay in diagnosis is associated with high morbidity
and mortality rate after bowel injury, especially with small
bowel perforation. Exploratory laparoscopy or laparotomy
followed by repair via suture or resection and anastomosis is
essential [13, 15]. In one of the earlier reported cases of bowel
perforation during microscopic discectomy, the patient was
not diagnosed until after 48 hours and the delay resulted in
peritonitis, sepsis, and death of the patient [16]. In our case,
the surgical team was consulted within one postoperative day
and the patient was rapidly diagnosed which prevented her
from developing septic shock and a potentially fatal outcome. (Figure 2(b)). With very light manipulation, enteric contents
exuded from the small bowel and the procedure was con-
verted to an open exploratory laparotomy. After repairing the
enterotomy with interrupted Vicryl sutures and reinforcing it
with silk suture in a Lembert fashion, the abdomen was irri-
gated copiously. Other than the jejunal-ileal perforation, no
other intra-abdominal pathologies were noted during explo-
ration. The patient’s postoperative hospital course was unre-
markable and she tolerated the small bowel repair without
complication. Neurosurgical intervention in the vertebral column will
remain common as long as chronic low back pain is a leading
chief complaint in the United States. Authors’ Contribution All authors contributed to evaluating and/or managing the
case and to writing/editing the paper. R. H. Krieger is the
paper guarantor. Conflict of Interests The authors declare that there is no conflict of interests
regarding the publication of this paper. 3. Discussion When considering the supportive anatomy of the vertebral
column, specifically the annulus fibrosus and the anterior
longitudinal ligament, it is not inconceivable that ventral
perforation is a rather rare complication of laminectomy. In
a study of 30,000 lumbar discectomies, there was a reported
ventral perforation rate of 0.016% [8] and when reviewing
another study of documented cases it appears that this
would be even less common during a laminectomy [7]. This
seems logical that laminectomies would be a rarer cause of
anterior perforation as the lamina is located dorsally on the
vertebral body and the bowel and retroperitoneal vasculature
lies anterior to the vertebral body. The most acute, life-
threatening complication from an anterior perforation would
be due to an intraoperative vascular injury, which may be
evident by brisk bleeding, hypotension, or shock. In fact,
hemorrhage of the large retroperitoneal vessels is the most
common complication due to anterior perforation of the ver-
tebral column [9–11]. Due to the pressure in the common iliac
arteries, the most commonly injured vessels during lumbar
laminectomy or discectomy, an acute vascular injury is likely
to be quickly recognized by the surgical team. However, in
the few reported cases of small bowel injury, the patients
will typically begin experiencing severe abdominal pain by
the second postoperative day, which was consistent with our
patient’s hospital course [12]. Therefore, it is imperative to
have a high index of suspicion for bowel perforation when
a postoperative laminectomy or discectomy patient develops
peritoneal signs.h Consent Informed patient consent was obtained and all patient iden-
tifiers have been removed. 2. Case Report While it is not a typical
complication, perforation of the viscus is associated with a
high mortality rate; therefore, the surgeon must maintain a
high index of suspicion when a patient presents with an acute
abdomen after lumbar spinal surgery and be prepared to
emergently surgically diagnose and correct the injury in the
operating room. References [1] R. A. Deyo and Y. J. Tsui-Wu, “Descriptive epidemiology of low-
back pain and its related medical care in the United States,”
Spine, vol. 12, no. 3, pp. 264–268, 1987.h p
g
The incidence of intestinal injury following lumbar dis-
cectomy was reported, in a large-scale study with 68,329
patients, to be 0.0015% by the German Society of Neurolog-
ical Surgery [8]. Thorough literature search identified this as
the 16th reported case of small bowel injury occurring after a
discectomy or laminectomy, dating back to the first reported
case by Harbison in 1954 [7, 13, 14]. Of all of the cases discov-
ered in the review of the literature, we believe that this is the
2nd reported case of small bowel injury after a laminectomy
[7]. The most likely mechanism by which the small bowel may
be penetrated is due to the root of the mesentery arising from
the anterior vertebral column at approximately L2, traveling
obliquely and terminating at the right sacroiliac joint. When
the patient is prone during the operation, segments of
the small bowel may appear anterior to the lumbar vertebral
column [6]. This mechanism, along with deep surgical
penetration during laminectomy, is precisely the means by
which our patient had her jejunal-ileal junction perforated. [2] J. D. Cassidy, L. J. Carroll, and P. Cˆot´e, “The Saskatchewan health
and back pain survey: the prevalence of low back pain and
related disability in Saskatchewan adults,” Spine, vol. 23, no. 17,
pp. 1860–1866, 1998. [3] L. Kalichman, R. Cole, D. H. Kim et al., “Spinal stenosis preva-
lence and association with symptoms: the Framingham Study,”
Spine Journal, vol. 9, no. 7, pp. 545–550, 2009. [4] T. Amundsen, H. Weber, H. J. Nordal, B. Magnaes, M. Abdel-
noor, and F. Lille˚as, “Lumbar spinal stenosis: conservative or
surgical management? A prospective 10-year study,” Spine, vol. 25, no. 11, pp. 1424–1436, 2000. [5] Y. Chang, D. E. Singer, Y. A. Wu, R. B. Keller, and S. J. Atlas, “The
effect of surgical and nonsurgical treatment on longitudinal
outcomes of lumbar spinal stenosis over 10 years,” Journal of the
American Geriatrics Society, vol. 53, no. 5, pp. 785–792, 2005. [6] P. Hoff-Olsen and J. Wiberg, “Small bowel perforation as a com-
plication of microsurgical lumbar diskectomy: a case report and
brief review of the literature,” The American Journal of Forensic
Medicine & Pathology, vol. 22, no. 3, pp. 319–321, 2001. References Case Reports in Surgery 4 [7] M. J. Cases-Bald´o, V. Soria-Aledo, J. A. Miguel-Perello, J. L. Aguayo-Albasini, and M. R. Hern´andez, “Unnoticed small
bowel perforation as a complication of lumbar discectomy,”
Spine Journal, vol. 11, no. 1, pp. e5–e8, 2011. [8] L. F. Ramirez, R. Thisted, G. W. Sypert, and N. Horwitz, “Com-
plications and demographic characteristics of patients undergo-
ing lumbar discectomy in community hospitals,” Neurosurgery,
vol. 25, no. 2, pp. 226–231, 1989. [9] R. Goodkin and L. L. Laska, “Vascular and visceral injuries
associated with lumbar disc surgery: medicolegal implications,”
Surgical Neurology, vol. 49, no. 4, pp. 358–372, 1998. [10] Y.-D. Tsai, P.-C. Yu, T.-C. Lee, H.-S. Chen, S.-H. Wang, and
Y.-L. Kuo, “Superior rectal artery injury following lumbar disc
surgery,” Journal of Neurosurgery, vol. 95, no. 1, pp. 108–110, 2001. [11] S. Papadoulas, D. Konstantinou, H. P. Kourea, N. Kritikos, N. Haftouras, and J. A. Tsolakis, “Vascular injury complicating
lumbar disc surgery. A systematic review,” European Journal of
Vascular and Endovascular Surgery, vol. 24, no. 3, pp. 189–195,
2002. [12] J. K. Houten, A. K. Frempong-Boadu, and M. S. Arkovitz,
“Bowel injury as a complication of microdiscectomy: case
report and literature review,” Journal of Spinal Disorders and
Techniques, vol. 17, no. 3, pp. 248–250, 2004. [13] D.-S. Kim, J.-K. Lee, K.-S. Moon, J.-K. Ju, and S.-H. Kim, “Small
bowel injury as a complication of lumbar microdiscectomy: case
report and literature review,” Journal of Korean Neurosurgical
Society, vol. 47, no. 3, pp. 224–227, 2010. [14] S. P. Harbison, “Major vascular complications of intervertebral
disc surgery,” Annals of Surgery, vol. 140, no. 3, pp. 342–348,
1954. [15] J. M. Dixon, A. B. Lumsden, and J. Piris, “Small bowel perfora-
tion,” The Journal of the Royal College of Surgeons of Edinburgh,
vol. 30, no. 1, pp. 43–46, 1985. [16] I. W. Birkeland Jr. and T. K. F. Taylor, “Bowel injuries coincident
to lumbar disk surgery: a report of four cases and a review of the
literature,” Journal of Trauma, vol. 10, no. 2, pp. 163–168, 1970.
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Askeri Okul Yetenek Sınavlarına Hazırlanan Bireylerin Yaratıcılık Algı Düzeylerinin İncelenmesi
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DergiPark (Istanbul University)
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Investigation of Creativity Perception Levels of Individuals Prepared for
Military School Aptitude Exams Abstract Article Info
Received: 13.10.2021
Accepted: 27.12.2021
Online Published:
27.12.2021 Creativity is the ability to apperceptive an issue clearly by using one’s imagination and
then present an opinion or concept in relation to that. This study, it is aimed to examine
the individual creativity perception levels of individuals preparing for military school
exams in terms of some variables (age, gender, marital status, education level). The study
consist of 204 people who preparing for military school exam in physical education and
sports preparation courses. The Personal Information Form and Individual Creativity
Factor which is sub-factor of the Organizational creativity scale created by the researcher
was used as a data collection tool in the study. SPSS 22 software package used in the
analysis of the study. The total mean score of the scale items was checked fort he analysis. Independent Sample t-Test was performed to determine among which groups was the
difference. While perform analysis, the eror margin was taken as p<0,05. In the analyzes,
it was observed that there was no significant difference in the individual creativity
perception levels of the individuals preparing for the military school exams in terms of
age, gender, marital status and education level variables. As a result, it was determined
that the individual creativity perception levels of the individuals preparing for the military
school exams were at a sufficient level and the variables were not effective on the
individual creativity perception levels. 1 Fırat Üniversitesi, Spor Bilimleri Fakültesi, Elazığ /Türkiye. Yayın Bilgisi
Gönderi Tarihi: 13.10.2021
Kabul Tarihi: 27.12.2021
Online Yayın Tarihi:
27.12.2021 Özet Yaratıcılık, kişinin hayal gücünü kullanarak bir sorunu net bir şekilde kavrayabilmesi ve
devamında bununla ilgili olarak bir görüş veya kavram ortaya koyabilmesidir. Bundan
dolayı bu çalışmada, askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı
düzeylerinin bazı değişkenler (yaş, cinsiyet, medeni durum, eğitim düzeyi) bakımından
incelenmesi amaçlanmıştır. Çalışmaya özel yetenek sınavlarına hazırlık kurslarında
askeri okul yetenek sınavına hazırlanan 204 kişi katılmıştır. Çalışmada veri toplama aracı
olarak araştırmacı tarafından oluşturulan Kişisel Bilgi Formu ve Örgütsel Yaratıcılık
Ölçeği (ÖLÖ)’ nin alt faktörü olan Bireysel Yaratıcılık faktörü kullanılmıştır. Elde edilen
verilerin analizi için SPSS 22 istatistik programından yararlanılmıştır. Verilerin analizi
için ölçek maddelerinin toplam puan ortalamasına bakılmıştır. Farklılığın hangi gruplar
arasında olduğunu tespit etmek için Independent Sample T-Testi yapılmıştır. Analizler
yapılırken hata payı p<0,05 olarak alınmıştır. Analizlerlede, askeri okul sınavlarına
hazırlanan bireylerin bireysel yaratıcılık algı düzeylerinde yaş, cinsiyet, medeni durum
ve eğitim düzeyi değişkenleri açısından anlamlı fark olmadığı görülmüştür. Sonuç olarak
askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeylerinin yeterli
düzeyde olduğu ve değişkenlerin bireysel yaratıcılık algı düzeyleri üzerinde etkili
olmadığı tespit belirlenmiştir. Yayın Bilgisi
Gönderi Tarihi: 13.10.2021
Kabul Tarihi: 27.12.2021
Online Yayın Tarihi:
27.12.2021 Anahtar Kelimeler
Beğenilme, İnternet,
Öğrenci, Sosyal Ağlar,
Sosyal Medya. Investigation of Creativity Perception Levels of Individuals Prepared for
Military School Aptitude Exams Spor Eğitim Dergisi
http://dergipark.gov.tr/seder
Cilt 5, Sayı 3, 171-177 (2021) Spor Eğitim Dergisi
http://dergipark.gov.tr/seder
Cilt 5, Sayı 3, 171-177 (2021) Anahtar Kelimeler
Beğenilme, İnternet,
Öğrenci, Sosyal Ağlar,
Sosyal Medya. Keywords
Internet, Likes, Student,
Social Media, Social
Networks. 1 Fırat Üniversitesi, Spor Bilimleri Fakültesi, Elazığ /Türkiye. Özgün Makale Özgün Makale Çetin TAN1, Muhammed GÜLER1, Mesut BULUT1 Araştırma Grubu Araştırmanın evrenini Elazığ ili Lider Beden Eğitimi ve Hazırlık Kursu’ nda askeri okul sınavlarına
hazırlanan 296 öğrenci oluştururken aralarından rastgele örneklem yöntemi ile seçilen 204 kişi örneklem
grubunu oluşturmuştur (Ural ve Kılıç, 2006). Giriş Bireyler, yaratıcılığın gelişimine kaynaklık etmektedirler. Bireysel yaratıcılığı geliştirmek için
bireyleri güdülemek lazımdır. Örgüt ortamında güdülemeyi meydana getirmek için kullanılacak temel öğe,
yöneticilerin davranış şekilleri ve örgüt iklimidir. Kısaca örgütün sahip olduğu kültürel kaynaklardır. Bu 171 Spor Eğitim Dergisi, 2021, Cilt 5, Sayı 3, 171-177 Ç. Tan, M. Güler, M. Bulut açıdan, örgütsel yaratıcılığa ulaşmak için ilk olarak grubun ve bireyin nasıl kişilik özelliklerine sahip olması
gerektiği ortaya konulmalıdır. Yaratıcılık, farklı alanlarda ve farklı yoğunlukta olmak koşulu ile bütün
insanlarda mevcut bir özelliktir. Bundan dolayı, net bir şekilde bazı insanlar yaratıcılık özelliklerine sahiptir,
bazı insanlar ise yaratıcılık özelliklerine sahip değildir denemez. Bütün insanlar az çok yaratıcılık özellikleri
gösterebilirler. Yaratıcılık özelliği bir anda ortaya çıkar gibi görünse de gerçekte birikim yoluyla oluşan bir
durumdur. Yaratıcılık, bireyde öğrenme kabiliyetleri içinde geliştirilmesi en zor olan, ancak aynı zamanda
en önemli unsurdur (Hacımustafaoğlu, 2008). Yaratıcılık özelliği, yalnızca şanslı bir azınlığa tanınmıştır. İlham veren ve kontrolü sağlanamayan
bir güç olduğu düşünülmektedir. Yaratıcılık ve iş hayatı birbirinden rahatsızlık duyan iki yakın arkadaş
gibidirler. Yaratıcılığı bir köşeye itip, konuyu alanında iddialı bireylere bırakmak “işe yaramışsa herhangi
bir değişikliğe ihtiyaç yok” düşüncesini benimsemek ve geçmiş başarıları taklit etmek, bu şekilde işimizde
ilerlemeye çalışmak bir yanlıştır (Akdeğirmen, 2015). Yaratıcılık ile alakalı yapılan çalışmaların çoğunun hedef noktası, yaratıcılık özelliği olan bireyin
belirlenmesine yönelik olduğu gözlenmektedir. Yaratıcılık konusu hakkında çalışan araştırmacılar, bireyi
yaratıcı kılan ve insanlardan ayıran özelliklerinin neler olduğunu bulmak için birçok çalışma yapmışlardır. Yaratıcılık özelliği olan kişiye odaklanan çalışmalar, yaratıcı bireyi öteki insanlardan ayıran birtakım
özelliklerinin bulunduğunu tespit etmişlerdir. Ancak bireyi yaratıcı yapan en önemli özellik onun içsel
motivasyonu olduğu son dönemlerin çok önemli bir tespitidir (Pıçakçı, 2013). Yaratıcılık, entelektüel bilgi birikimini, kişisel özgürlüğün hakim olduğu bir kişilik yapısını, yaşamı
algılama, aktarma yetisini ve sezgisini içeren ve çok boyutla açıklanan bir kavramdır. İnsanların kişilik
özellikleri, tecrübeleri, kişisel yetenekleri, sorunlarla başa çıkma becerileri ve motivasyonları, bireysel
açıdan yaratıcılığı etkileyen unsurlardır. Yaratıcılık, insan zekâsının temel bir özelliğidir. Yaratıcı bir fikir,
şaşırtıcı, yeni ve sonuçları açısından değerli olan bir düşüncedir. Bireysel yaratıclık insanların hayatlarına
yön veren bir olgudur (Sunar, 2020). Bu bireysel yaratıcılık sayesinde insanlar kendi iş imkânlarını sağlamaktadırlar. Bu anlamda
bireylerde öz güven, toplumsal sosyalleşme bu bağlamda ön plana çıkmaktadır. Bireysel yaratıcılık ile
özellikle özel yetenek sınavlarında ön plana çıkmaktadır. Bu sınavlarda bireylerin kendi çabalarıyla yani
yaratıclıkları ile bu sınavda başarılı olabileceklerdir. Askeri okul yetenek sınavlarında bireylerin bireysel yaratıcılık algı düzeyleri nedir? Giriş Askeri okul
yetenek sınavlarına katılan bireylerin algı düzeyleri arasında değişkenlere göre fark varmıdır? Verilerin Toplanması Çalışmamızda veri toplama aracı olarak anket formu kullanılmıştır. Kullanılan anket formu iki
kısımdan meydana gelmektedir. Birinci kısımda araştırmacılar tarafından oluşturulan kişisel bilgi formu (‘1. 172 Spor Eğitim Dergisi, 2021, Cilt 5, Sayı 3, 171-177 Ç. Tan, M. Güler, M. Bulut Cinsiyet’, ‘2. Yaş’, ‘3. Medeni durum’, ‘4. Eğitim Durumu’), ikinci kısımda ise Balay (2010)’ ın geliştirmiş
olduğu Örgütsel Yaratıcılık Ölçeği (ÖYÖ)’ nin 3 faktöründen biri olan Bireysel Yaratıcılık Faktörü
kullanılmıştır. Örgütsel Yaratıcılık Ölçeği (ÖYÖ) Balay (2010) tarafından geliştirilen ölçek bireysel yaratıcılık, örgütsel yaratıcılık ve toplumsal
yaratıcılık olmak üzere 3 faktörden oluşmaktadır. Çalışmamızda Örgütsel Yaratıcılık Ölçeği (ÖYÖ)’ nin
Bireysel Yaratıcılık Faktörü kullanılmıştır. Bireysel Yaratıcılık Faktörü 16 madde ve 5 likertten meydana
gelmektedir (Balay, 2010). Verilerin Analizi Verileri analiz etmek için SPSS 22 istatistik programından yararlanılmıştır. Askeri okul sınavlarına
hazırlanan bireylerin; bireysel yaratıcılık algı düzeylerini tespit etmek amacıyla “aritmetik ortalama (X) ve
standart sapma (Ss)” değerlerine bakılmıştır. Askeri okullara hazırlanan öğrencilerin yaş, cinsiyet, medeni
durum ve eğitim düzeyine göre anlamlı farklılık gösterip göstermediğini tespit etmek için, aritmetik
ortalamalarına bakılmış ve independent sample t testi yapılmıştır. Analizler yapılırken anlamlık düzeyi
p<0,05 olarak alınmıştır. Bireysel yaratıcılık algı düzeylerine ilişkin verilerin ortalamalara göre
yorumlanması ve ortalamanın hangi düzeye girdiğini belirlemek amacıyla bu dereceler araştırmacılar
tarafından 3 düzeyde toplanmıştır. Bu düzeyler “tam katılıyorum ve çok katılıyorum (yeterli düzey)”, “orta
derecede katılıyorum (orta düzey)” ve “az katılıyorum ve hiç katılmıyorum (yetersiz düzey)” olarak
belirlenmiştir (Tablo 1). Tablo 1. Bireysel Yaratıcılık Algı Düzeyine İlişkin Sınırlar ve Gruplamalar
Seçenekler
Sınırlar
Düzeyler
Sınırlar
Kesinlikle katılıyorum
(5)
4,20-5,00
Yeterli Düzey
3,40-5,00
Katılıyorum
(4)
3,40-4,19
Kararsızım
(3)
2,60-3,39
Orta Düzey
2,60-3,39
Katılmıyorum
(2)
1,80-2,59
Yetersiz Düzey
1,00-2,59
Kesinlikle katılmıyorum
(1)
1,00-1,79
Bulgular
Tablo 2. Bağımsız Değişkenler Tablosu
Değişkenler
Grup
N
%
Cinsiyet
Erkek
64
31,40
Kadın
140
68,60
Yaş
20-24 Arası
117
57,40
25-29 Arası
87
42,60
Medeni Durum
Evli
34
16,70
Bekâr
170
83,30
Eğitim Düzeyi
Ön Lisans
97
47,50
Lisans ve Üstü
107
52,50 Tablo 1. Bireysel Yaratıcılık Algı Düzeyine İlişkin Sınırlar ve Gruplamalar
Seçenekler
Sınırlar
Düzeyler
Sınırlar
Kesinlikle katılıyorum
(5)
4,20-5,00
Yeterli Düzey
3,40-5,00
Katılıyorum
(4)
3,40-4,19
Kararsızım
(3)
2,60-3,39
Orta Düzey
2,60-3,39
Katılmıyorum
(2)
1,80-2,59
Yetersiz Düzey
1,00-2,59
Kesinlikle katılmıyorum
(1)
1,00-1,79 Tablo 1. Bireysel Yaratıcılık Algı Düzeyine İlişkin Sınırlar ve Gruplamalar Bulgular Bulgular
Tablo 2. Bağımsız Değişkenler Tablosu
Değişkenler
Grup
N
%
Cinsiyet
Erkek
64
31,40
Kadın
140
68,60
Yaş
20-24 Arası
117
57,40
25-29 Arası
87
42,60
Medeni Durum
Evli
34
16,70
Bekâr
170
83,30
Eğitim Düzeyi
Ön Lisans
97
47,50
Lisans ve Üstü
107
52,50 173 Spor Eğitim Dergisi, 2021, Cilt 5, Sayı 3, 171-177 Ç. Tan, M. Güler, M. Bulut Askeri okul sınavlarına hazırlanan bireylerin çoğunluğu (% 68,60) kadın, yarısından fazlası (%
57,40) 20-24 yaşlar arasında, %52,50 lisans ve üstü mezunu ve büyük çoğunluğu (%83,30) bekâr olarak
görülmüştür. Tablo 3. Askeri Okul Sınavlarına Hazırlık Öğrencilerinin Bireysel Yaratıcılık Algı Düzeyleri Tablo 3. Askeri Okul Sınavlarına Hazırlık Öğrencilerinin Bireysel Yaratıcılık Algı Düzeyleri
Örgütsel Yaratıcılık Ölçeğinin Bireysel Yaratıcılık Faktörü Maddelerinin Toplam Puan
Ortalaması
Ss
3,97
0,83
Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algıları “yeterli düzey (
= 3,71-
x
x Örgütsel Yaratıcılık Ölçeğinin Bireysel Yaratıcılık Faktörü Maddelerinin Toplam Puan
Ortalaması
Ss
3,97
0,83
x Örgütsel Yaratıcılık Ölçeğinin Bireysel Yaratıcılık Faktörü Maddelerinin Toplam Puan
Ortalaması
Ss
3,97
0,83
x Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algıları “yeterli düzey (
= 3,71-
4,14)” sınırları içerisindedir. x Tablo 4. Bireysel Yaratıcılık Algı Düzeyi Yaş Değişkenine Göre t- Testi
Yaş
N
ss
t
p
20-24 Arası
117
3,96
0,86
-,240
,810
25-29 Arası
87
3,99
0,79
x Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeyleri yaş değişkeni
açısından incelendiğinde 20-24 yaş arasında olanların ortalamasının
=3,96, 25-29 yaşlar arasında olanların
ortalamasının ise
=3,99 olduğu belirlenmiştir. Belirlenen farklılaşmanın anlamlı olup olmadığını test
etmek amacıyla t-testi uygulanmış ve farkın istatistiki açıdan anlamlı olmadığı saptanmıştır (p>,05). x
x Tablo 5. Bireysel Yaraticilik Algı Düzeyi Cinsiyet Değişkenine Göre T- Testi
Cinsiyet
N
ss
t
p
Erkek
64
4,00
0,85
,330
,730
Kadın
140
3,96
0,83
x Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeyleri cinsiyet değişkeni
açısından incelendiğinde erkeklerin ortalamasının
= 4,00 kadınların ortalamasının ise
= 3,96 olduğu
belirlenmiştir. Belirlenen farklılaşmanın anlamlı olup olmadığını test etmek amacıyla t-testi uygulanmış ve
farkın istatistiki açıdan anlamlı olmadığı saptanmıştır (p>,005). x
x Tablo 6. Bireysel Yaratıcılık Algı Düzeyi Medeni Durum Değişkenine Göre t- Testi
Medeni Durum
N
ss
t
p
Evli
34
4,02
0,87
,360
,710
Bekar
170
3,96
0,83
x Tablo 6. Bireysel Yaratıcılık Algı Düzeyi Medeni Durum Değişkenine Göre t- Testi Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeyleri medeni durum
değişkeni açısından incelendiğinde evlilerin ortalamasının
=4,02 bekârların ortalamasının ise
=3,96
olduğu belirlenmiş. Bulgular Belirlenen farklılaşmanın anlamlı olup olmadığını test etmek amacıyla t-testi uygulanmış
ve farkın istatistiki açıdan anlamlı olmadığı saptanmıştır (p>,005). x
x Tablo 7. Bireysel Yaratıcılık Algı Düzeyi Eğitim Düzeyi Değişkenine Göre t- Testi
Eğitim Düzeyi
N
Ss
t
p
Ön Lisans
97
4,06
,79
1,370
,170
Lisans ve Üstü
107
3,90
,86
Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeyleri eğitim düzeyi
değişkeni açısından incelendiğinde ön lisans mezunlarının ortalamasının
=4,06 lisans ve üstü mezunların
x
x Tablo 7. Bireysel Yaratıcılık Algı Düzeyi Eğitim Düzeyi Değişkenine Göre t- Testi
Eğitim Düzeyi
N
Ss
t
p
Ön Lisans
97
4,06
,79
1,370
,170
Lisans ve Üstü
107
3,90
,86
Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeyleri eğitim düzeyi
değişkeni açısından incelendiğinde ön lisans mezunlarının ortalamasının
=4,06 lisans ve üstü mezunların
x
x Tablo 7. Bireysel Yaratıcılık Algı Düzeyi Eğitim Düzeyi Değişkenine Göre t- Testi
Eğitim Düzeyi
N
Ss
t
p
Ön Lisans
97
4,06
,79
1,370
,170
Lisans ve Üstü
107
3,90
,86
x Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeyleri eğitim düzeyi
değişkeni açısından incelendiğinde ön lisans mezunlarının ortalamasının
=4,06 lisans ve üstü mezunların
x Askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeyleri eğitim düzeyi
değişkeni açısından incelendiğinde ön lisans mezunlarının ortalamasının
=4,06 lisans ve üstü mezunların
x 174 Spor Eğitim Dergisi, 2021, Cilt 5, Sayı 3, 171-177 Ç. Tan, M. Güler, M. Bulut ortalamasının ise
=3,90 olduğu belirlenmiştir. Belirlenen farklılaşmanın anlamlı olup olmadığını test
etmek amacıyla t-testi uygulanmış ve farkın istatistiki açıdan anlamlı olmadığı saptanmıştır (p>,005). x Tartışma ve Sonuç Bu çalışmanın amacı, beden eğitimi ve spor hazırlık kurslarında askeri okul sınavlarına hazırlanan
bireylerin bireysel yaratıcılık algı düzeylerini tespit etmektir. Aynı zamanda askeri okul sınavlarına
hazırlanan öğrencilerin cinsiyete, yaşa, eğitim düzeyine ve medeni duruma göre bireysel yaratıcılık
algılarının farklılık gösterip göstermediğini tespit etmektir. Çalışmamıza beden eğitimi ve spor hazırlık kurslarında, askeri okul sınavlarına hazırlanan 204 kişi
katılmıştır. Katılımcılar; 20- 24 yaş 117 kişi, 25- 29 yaş 87 kişiden oluşmuştur. Çalışmaya 64 erkek 140 kadın
katılmıştır. Katılımcıların 34’ ü evli, 170’ i bekârdır. Katılımcılardan 97’ si ön lisans mezunu iken 107’ si
lisans ve lisans üstü eğitim seviyesine sahiptir. Çalışmada yapılan analizler sonucu, askeri sınavlara hazırlanan öğrencilerin bireysel yaratıcılık algı
düzeyleri (= 3.97) yeterli seviyede görülmüştür. Çalışmamızda askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeylerinin yaş
değişkenine göre yaptığımız t testi sonuçlarına baktığımızda gruplar arasında herhangi bir anlamlı fark
görülmemiştir. Bunun nedeninin, ön lisans ve lisans derecesinde eğitimini tamamlayan bireylerin askeri
okullara girebilme üst yaş sınırının 30 yaş olması ve 30 yaş üstü bireylerin çalışmaya katılmamış olmasından
kaynaklandığı düşünülmektedir. Yahşi ve Özbek (2014)’ in yaptıkları çalışmaya baktığımızda yaş
değişkenine göre herhangi bir anlamlı farka rastlanmamıştır. Bu çalışmanın bulguları bizim bulgularımız ile
örtüşmektedir. Çalışmamızda askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeylerinin
cinsiyet değişkenine göre yaptığımız t testi sonuçlarına baktığımızda gruplar arasında herhangi bir anlamlı
fark görülmemiştir. Yenice ve Yavaşoğlu (2018) tarafından fen bilgisi öğretmen adaylarının bireysel
yaratıcılık algı düzeylerini incelemek amacıyla yapılan çalışmayı incelediğimizde de kadınlar ve erkekler
arasında herhangi bir fark görülmemiştir. Güngör ve ark. (2018) tarafından yapılan çalışmada da cinsiyet
değişkeninde gruplar arasında herhangi bir anlamlı fark görülmemiştir. Canal (2017)’ ın yapmış olduğu
örgütsel özdeşleşmenin bireysel yaratıcılık üzerindeki etkisinin incelendiği çalışmada da kadın ve erkekler
arasında anlamlı bir fark tespit edilmemiştir. Bu çalışmalardan görüldüğü gibi bireysel yaratıcılık algı
düzeylerinde cinsiyet değişkeni açısından istatistiki fark tespit edilmemiştir. Bu çalışmalar çalışmamızı
destekler nitelikte sonuçlardan oluşmaktadır. Bu veriler ışığında kadınlar ve erkeklerin bireysel yaratıcılık
algı düzeylerinin birbirine yakın olduğunu söyleyebiliriz. Çalışmamızda askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeylerinin
medeni durum değişkenine göre yaptığımız t testi sonuçlarına baktığımızda gruplar arasında herhangi bir
anlamlı fark görülmemiştir. Aynı şekilde Yahşi ve Özbek (2014)’ in yapmış oldukları çalışmada da gruplar
arasında anlamlı bir fark görülmemiştir. Bulgular sonucunda; askeri okul sınavlarına hazırlanan bireylerin
bireysel yaratıcılık algı düzeylerinin evli ya da bekâr olma durumu ile ilişkili olmadığını söyleyebiliriz. 175 Spor Eğitim Dergisi, 2021, Cilt 5, Sayı 3, 171-177 Ç. Tan, M. Güler, M. Öneriler •
Bu çalışmanın neticesinde Askeri okul yetenek sınalarına hazırlanan bireylerin algı düzeyleri
hakkında bundan sonraki yapılacak çalışmlara yol gösterici olabilir. •
Bu çalışmanın neticesinde Askeri okul yetenek sınalarına hazırlanan bireylerin algı düzeyleri
hakkında bundan sonraki yapılacak çalışmlara yol gösterici olabilir. •
Yaptığımız çalışma ile kişilerin kendi yaratcılıklarının farkına vararak bundan sonraki gireceklerdeki
sınavlarda daha bilinçli ve daha başarılı olacakları düşünülmektedir. •
Yaptığımız çalışma ile kişilerin kendi yaratcılıklarının farkına vararak bundan sonraki gireceklerdeki
sınavlarda daha bilinçli ve daha başarılı olacakları düşünülmektedir. •
Yaptığımız çalışma ile kişilerin kendi yaratcılıklarının farkına vararak bundan sonraki gireceklerdeki
sınavlarda daha bilinçli ve daha başarılı olacakları düşünülmektedir. Ural, A. ve Kılıç, İ. (2006). Bilimsel Araştırma Süreci ve Spss ile Veri Analizi (2. baskı). Ankara: Detay
Yayıncılık. Tartışma ve Sonuç Bulut Çalışmamızda askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeylerinin
eğitim düzeyi değişkenine göre yaptığımız t testi sonuçlarına baktığımızda gruplar arasında herhangi bir
anlamlı fark görülmemiştir. Askeri okul sınavlarına hazırlanan ön lisans seviyesinde eğitim almış olma ile
lisans ve üstü seviyesinde eğitim almış olmanın bireylerin bireysel yaratıcılık algı düzeylerine herhangi bir
etkisinin olmadığını söyleyebiliriz. Konuyla ilgili olarak yapılan literatür taraması sonucunda eğitim durumu
ile ilgili herhangi bir veriye rastlanmamıştır. Çalışmamızın bundan sonra yapılacak olan çalışmalarda kaynak
olarak kullanılabileceği düşünülmektedir. Sonuç olarak; askeri okul sınavlarına hazırlanan bireylerin bireysel yaratıcılık algı düzeylerinin
yeterli seviyede olduğu görülmüştür. Bireysel yaratıcılık algı düzeylerinin cinsiyet, yaş, medeni durum ve
eğitim durumu gibi değişkenlere göre farklılaşmadığı tespit edilmiştir. Bu çalışmanın bundan sonra yapılacak
olan çalışmalara kaynak olabileceği düşünülmektedir. Kaynaklar Akdeğirmen, E. (2015). Sosyal Medya Kullanımının İnovasyon, Bireysel Yaratıcılık ve Çalışan
Performansına Etkisi. Yüksek Lisans Tezi. İstanbul Arel Üniversitesi, Sosyal Bilimler Enstitüsü. İşletme Anabilim Dalı, İstanbul. Akdeğirmen, E. (2015). Sosyal Medya Kullanımının İnovasyon, Bireysel Yaratıcılık ve Çalışan
Performansına Etkisi. Yüksek Lisans Tezi. İstanbul Arel Üniversitesi, Sosyal Bilimler Enstitüsü. İşletme Anabilim Dalı, İstanbul. Balay, R. (2010). Öğretim elemanlarının örgütsel yaratıcılık algıları. Ankara Üniversitesi Eğitim Bilimleri
Fakültesi Dergisi, 43(1), 41-78. Canal, B. (2017). Örgütsel Özdeşleşmenin Bireysel Yaratıcılık Üzerindeki Etkisi: Bankacılık Sektöründe Bir
Araştırma. Nevşehir. Yüksek Lisans Tezi. Hacı Bektaş Veli Üniversitesi, Sosyal Bilimler Enstitüsü,
İşletme Anabilim Dalı, Nevşehir. Güngör, S., Küçün, N.T. ve Sezgin, M. (2018). Girişimci öğretim elemanlarının psikolojik sermaye ve
bireysel yaratıcılıklarının incelenmesi. Sosyal Bilimler Metinleri, 2, 50-64. Hacımustafaoğlu, M.F. (2008). Personel Güçlendirme Algılarının Bireysel Yaratıcılığa Etkisi ve Otel
İşletmelerinde Bir Uygulama. Yüksek Lisans Tezi. Marmara Üniversitesi, Sosyal Bilimler Enstitüsü,
İşletme Anabilim Dalı, Yönetim ve Organizasyon Bilim Dalı, İstanbul. Pıçakçı, K. (2013). Örgüt İkliminin Bireylerin Yaratıcılık Performansı Üzerindeki Etkileri: Moda Sektöründe
Bir Uygulama. Yüksek Lisans Tezi. Kocaeli Üniversitesi, Sosyal Bilimler Enstitüsü, İşletme
Anabilim Dalı. Yönetim ve Organizasyon Bilim Dalı, Kocaeli. Sunar, M.A. (2020). Duygusal Zekanın Bireysel Yaratıcılık Üzerindeki Etkisinde Örgütsel Özdeşleşmenin
Aracı Rolü. Yüksel Lisans Tezi. Artvin Çoruh Üniversitesi, Lisansüstü Eğitim Enstitüsü, İşletme
Anabilim Dalı, Artvin. Ural, A. ve Kılıç, İ. (2006). Bilimsel Araştırma Süreci ve Spss ile Veri Analizi (2. baskı). Ankara: Detay
Yayıncılık. 176 Spor Eğitim Dergisi, 2021, Cilt 5, Sayı 3, 171-177 Ç. Tan, M. Güler, M. Bulut Yahşi, Ü.Y. & Özbek, O.T.D. (2014). Gençlik ve Spor Bakanlığı Personelinin Örgüt İklimi Algıları ile
Örgütsel Yaratıcılık Düzeyleri. Doktora Tezi. Ankara Üniversitesi, Sağlık Bilimleri Enstitüsü Beden
Eğitimi ve Spor Anabilim Dalı, Ankara. Yahşi, Ü.Y. & Özbek, O.T.D. (2014). Gençlik ve Spor Bakanlığı Personelinin Örgüt İklimi Algıları ile
Örgütsel Yaratıcılık Düzeyleri. Doktora Tezi. Ankara Üniversitesi, Sağlık Bilimleri Enstitüsü Beden
Eğitimi ve Spor Anabilim Dalı, Ankara. Yenice, N. ve Yavaşoğlu, N. (2018). Fen bilgisi öğretmen adaylarının bireysel yenilikçilik düzeyleri ile
bireysel yaratıcılıkları arasındaki ilişkinin incelenmesi. Eğitimde Kuram ve Uygulama, 14(2), 107-
128. Yenice, N. ve Yavaşoğlu, N. (2018). Fen bilgisi öğretmen adaylarının bireysel yenilikçilik düzeyleri ile
bireysel yaratıcılıkları arasındaki ilişkinin incelenmesi. Eğitimde Kuram ve Uygulama, 14(2), 107-
128. Tan, Ç., Güler, M., & Bulut, M. (2021). Askeri Okul Yetenek Sınavlarına Hazırlanan Bireylerin Yaratıcılık
Algı Düzeylerinin İncelenmesi [Investigation of Creativity Perception Levels of Individuals Prepared for
Military School Aptitude Exams], Spor Eğitim Dergisi, 5 (3), 171-177. Makale Alıntısı Tan, Ç., Güler, M., & Bulut, M. (2021). Askeri Okul Yetenek Sınavlarına Hazırlanan Bireylerin Yaratıcılık
Algı Düzeylerinin İncelenmesi [Investigation of Creativity Perception Levels of Individuals Prepared for
Military School Aptitude Exams], Spor Eğitim Dergisi, 5 (3), 171-177. B 177 177
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Specijalna edukacija i rehabilitacija
(Beograd), Vol. 12, br. 3. 309-334, 2013. Specijalna edukacija i rehabilitacija
(Beograd), Vol. 12, br. 3. 309-334, 2013. Specijalna edukacija i rehabilitacija
(Beograd), Vol. 12, br. 3. 309-334, 2013. Joško SINDIK1
Institut za antropologiju, Zagreb, Hrvatska 1 E-mail: josko.sindik@inantro.hr KONSTRUKCIJA UPITNIKA STAVOVA ODGOJITELJICA
PREMA INKLUZIJI DJECE S TEŠKOĆAMA
U RAZVOJU U DJEČJE VRTIĆE Glavni cilj istraživanja bio je konstruirati mjerni instrument o
stavovima prema inkluziji za potrebe vrednovanja edukacije o inkluziji. Ispitan je uzorak odgojiteljica iz dječjih vrtića u Osijeku i Zagrebu, pri-
mjenom upitnika Stavovi stručnjaka prema inkluziji djece s teškoćama
u razvoju. Utvrdili smo postojanje sedam latentnih dimenzija stavova
prema inkluziji. Dimenzije stavova prema inkluziji, izvedene kao jed-
nostavne linearne kombinacije čestica koje definiraju pojedini faktor,
pokazuju nisku i granično zadovoljavajuću pouzdanost, koja varira od
vrlo niske (organizacijska prilagodba, kriteriji praćenja inkluzije) do
osrednje visoke. Od svih povezanosti između dimenzija stavova prema
inkluziji, više od trećine ih je većinom nisko, ali statistički značajno
i pozitivno povezanih. Na temelju utvrđenih metrijskih karakteristi-
ka, mjerni instrument može se smatrati dovoljno dobrim inicijalnim
polazištem za konstrukciju finalne verzije instrumenta namijenjenog
vrednovanju edukacije o inkluziji za odgojiteljice predškolske djece. Ključne riječi: djeca s teškoćama u razvoju, faktori stavova,
inkluzija, mjerni instrument, odgoj i obrazovanje UVOD Inkluzija je pokret protiv predrasuda kojim se želi razvijati osje-
ćaj društvene zajednice za toleranciju i razvoj pozitivnih stavova pre-
ma osobama koje su „drugačije” te kojim se teži pružiti podrška tim 309 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. osobama u procesu uključivanja u ravnopravno sudjelovanje u životu
ljudske zajednice. Stubbs (1998) smatra da je obrazovna inkluzija stra-
tegija čiji je krajnji cilj unaprjeđenje inkluzivnog društva u kojemu se
omogućuje svoj djeci/odraslima da u njemu sudjeluju i daju svoj do-
prinos, između ostalog i mogućnošću obrazovanja u skladu sa svojim
sposobnostima (bez obzira na spol, dob, sposobnost, etničku pripad-
nost ili razvojnu poteškoću). Posljednjih godina u Hrvatskoj u tijeku
su reforme školstva, a jedan od osnovnih principa reforme je nagla-
šavanje važnosti razvijanja jedinstvenog odgojno-obrazovnog sustava
za svu djecu, bez obzira na obilježja njihovog razvojnog procesa. Za
djecu s teškoćama može se reći da ona imaju trajnije (ili trajne) poseb-
ne potrebe. Naime, ove posebne potrebe odnose se na takva urođena
i stečena stanja organizma koja po svojoj prirodi zahtijevaju poseban
i prilagođen stručni pristup, usmjeren na omogućavanje razvoja sa-
čuvanih sposobnosti djeteta, što je preduvjet za djetetov kvalitetniji
daljnji odgoj i veću kvalitetu života (Daniels & Stafford, 2003). Inte-
gracija povećava mogućnost za sudjelovanje djeteta s teškoćama u ra-
zvoju u obrazovni sustav, dok je inkluzija puno sudjelovanje djeteta s
teškoćama u razvoju u obrazovni sustav određene ustanove (škole ili
dječjeg vrtića) (Stroeve, 1998). Integracija je dakle uključivanje djece s
teškoćama u razvoju u redovan sustav odgoja i obrazovanja, koja nuž-
no ne podrazumijeva prilagođavanje okruženja djetetu. Inkluzija je
pak proces kojim djeca iste kronološke dobi sa teškoćama i bez teškoća
u razvoju borave u istom okruženju, zbog zajedničke igre, druženja i
učenja: pojednostavljeno, to je proces učenja i odgajanja djece s teško-
ćama u razvoju zajedno s djecom koja nemaju takvih teškoća. Inkluzija
se može definirati kao praksa koja osigurava da sva djeca s teškoćama
sudjeluju s drugom djecom u svim aspektima programa dječjeg vrtića
(Smith & Luckasson, 1995), a odatle se o inkluzivnom obrazovanju
razmišlja kao o obrazovnom okruženju koje je sposobno da odgovori
na potrebe sve djece, bez obzira na njihove međusobne razlike. UVOD Pretpostavka je da u tom slučaju ta djeca imaju jednake mogućno-
sti u razvoju svojih tjelesnih, emocionalnih, društvenih i drugih spo- Pretpostavka je da u tom slučaju ta djeca imaju jednake mogućno-
sti u razvoju svojih tjelesnih, emocionalnih, društvenih i drugih spo-
sobnosti. Inkluzija djece s teškoćama u razvoju, zajedno s njihovim
vršnjacima koji nemaju tih teškoća, daje mogućnost svoj djeci da uče,
igraju se i žive zajedno, te da se razviju u osobe koje se razumiju i me-
đusobno poštuju (Leš, 2005). Djeca koja uče i žive u inkluzivnim gru- 310 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće pama, sukladno stupnju teškoća u razvoju, mogu maksimalno razviti
svoje potencijale te s drugima u društvu izmjenjivati svoje sposobno-
sti i znanja, višestruko doprinoseći poboljšanju uvjeta života u cijeloj
lokalnoj zajednici (Mustać i Vicić, 1996). Svaki dio tima sačinjenog od
roditelja i stručnjaka daje svoj doprinos uspješnosti i učinkovitosti
rada s djecom (Levandovski i Teodorović, 1991). Rad u inkluzivnim
grupama koristan je i djeci i obiteljima djece bez teškoća u razvoju, jer
u njima djeca lakše razvijaju nove odnose s različitom djecom i odra-
slima: uz bolje spoznavanje individualnih razlika među djecom, isto-
vremeno imaju mogućnost uvidjeti kako članovi obitelji, te odgojitelji
rade s djecom s teškoćama. Uspješno svladavanje iskušenja u ranijim
životnim fazama djetetova razvoja omogućuje roditeljima da im po-
stanu dobri skrbnici (Daniels & Stafford, 2003). f
Inkluzijom u širem smislu se smatraju odnosi na relaciji pojedi-
nac-društvo i obratno, te se ova vrsta inkluzije može nazvati socijalna
inkluzija. Tri su međuovisne dimenzije socijalne inkluzije: prostorna
(nestajanje socijalne i ekonomske udaljenosti); odnosna (osjećaj pri-
padanja i prihvaćanja; uključuje recipročnost i pozitivne interakcije,
željeti biti cijenjen, imati korisne društvene uloge; sudjelovati); funkci-
onalna (povećanje mogućnosti, sposobnosti, kompetencija) (Donnelly
& Coakley, 1991; Freiler, 2001). Svaka od navedenih dimenzija socijal-
ne inkluzije u kontekstu obrazovnog procesa može se smatrati inklu-
zijom u užem smislu (obrazovnom inkluzijom). j
j
Predškolski sustav kao početak „vertikale” cjelokupnog odgojno-
obrazovnog sustava u Hrvatskoj, zbog ustroja rada i fleksibilnosti pro-
grama uređenih legislativom ima najveću otvorenost prema realiziranju
inkluzije, ali se praktična provedba inkluzivnog obrazovanja redovito
problematizira (ne)davanjem mogućnosti djeci s teškoćama da se uklju-
če u redovne vrtiće. Problematizirajući teoriju inkluzivnog obrazovanja,
Primorac (prema Cerić, 2008) ističe važnost određivanja područja djelo-
vanja inkluzivnog obrazovanja. Potrebno je odvojiti inkluzivno obrazo-
vanje od problema uključenosti uopće. UVOD Inkluzija je termin koji ima uni-
verzalnu vrijednost i može se povezati s izjednačavanjem, nepristranim
tretmanom i pravima osobe da demokratski dijeli izvore u nekoj zemlji, i
to: političke, obrazovne, ekonomske i socijalne (Bezuk-Jakovina, 2002). Obrazovna inkluzija ovisi o edukacijskim i socijalnim vrijednostima kao
i osjetljivosti za individualnu vrijednost (Peck, Carlson & Helmstetter, 311 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. 1992). Odsustvo jasnih kriterija pri realiziranju inkluzivnog obrazova-
nja pomaže da svake godine redovni odgojno-obrazovni sustav napušta
ili ne može niti prihvatiti veliki broj djece koja imaju teškoće u razvoju. Djelotvorno inkluzivno obrazovanje ovisi i o odgojno-obrazovnom su-
stavu i o stavovima odraslih prema djetetu s teškoćama u razvoju te
stavovima prema inkluziji djeteta u redoviti sustav predškolskog odgoja
i obrazovanja (Peck, Carlson & Helmstetter, 1992). Među odraslima čiji su stavovi prema djetetu s teškoćama u ra-
zvoju te stavovima prema inkluziji posebno važni, izdvajaju se djete-
tovi roditelji i odgojitelji. Pozitivni stavovi roditelja prema djetetu po-
mažu formiranju kooperativnog, ljubaznog, emocionalno stabilnog,
iskrenog i pouzdanog djeteta (Daniels & Stafford, 2003), dok negativni
doprinose razvoju mahom nepoželjnih djetetovih karakteristika. Viši
socio-ekonomski status obitelji povezan je s ranijim utvrđivanjem po-
remećaja (ranim otkrivanjem i dijagnosticiranjem teškoća u razvoju),
te posljedično boljim mogućnostima za uspješniju stimulaciju razvoja
djeteta, pa se pokazalo primjerice da su obitelji s djetetom s manjim in-
telektualnim teškoćama često i nižeg ekonomskog statusa (Daniels &
Stafford, 2003). Viši socio-ekonomski status obitelji u prosjeku može
biti zaštitni faktor u svim dimenzijama socijalne inkluzije. Međutim,
kontradiktorno je da se češće zapošljavaju majke čija djeca nemaju teš-
koća/smetnji, dok obitelji s djetetom s teškoćama u razvoju u pravilu
imaju manje socijalne podrške (što utječe i na niži ekonomski status),
a upravo socijalna podrška pozitivno djeluje na ublažavanje stresa ko-
jemu su izloženi roditelji djece s teškoćama u razvoju (Bezuk-Jakovi-
na, 2002). Međutim, u ovom istraživanju istraživalo se prvenstveno
stavove odgojiteljica u dječjim vrtićima, koje su u suvremenom druš-
tvu u predškolskoj dobi djetetovi „drugi roditelji”. Istraživanja stavova odgojitelja prema inkluziji u praksi
predškolskog odgoja i obrazovanja Za dijete s teškoćama u razvoju koje pohađa redoviti dječji vrtić
iznimno je bitno da i odgojitelji te ostali stručnjaci u dječjim vrtićima
također imaju pozitivan i odgojno stimulativan stav prema djetetu s
teškoćama u razvoju. Nužan preduvjet je i pozitivan a realan stav pre-
ma inkluziji, te osposobljenost za inkluziju, koju nije lako i provesti u 312 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće praksi. Potrebno je oprezno i sustavno promisliti i definirati elemente
potrebne za realiziranje sustava podrške za rad u uvjetima inkluzi-
je (Kubelka i Sindik, 2009). Smjernice dijelom mogu dati i prethodna
istraživanja u ovom području, od kojih navodimo nekolicinu. Ispitujući stavove edukatora prema integraciji djece s teškoćama u
razvoju u redovni dječji vrtić, ispitani su stavovi odgojitelja uključenih u
programe inkluzije djece s teškoćama u razvoju u redovne predškolske
programe (Peck, Carlson & Helmstertter, 1992). Pokazalo se da odgo-
jitelji vjeruju da je uključivanje djece s teškoćama u razvoju u redovne
odgojne grupe pozitivno iskustvo, korisno za razvoj ostale djece (uz ono
koje je u inkluziji), te da djeca razvijaju i povoljniji self-koncept. Na razi-
ni SAD je provedeno istraživanje (Rose & Smith, 1993) kojim je ispitano
u kojoj se mjeri u stavovima edukatora percipiraju prepreke inkluzivnih
programa u ranom djetinjstvu. Ispitani su odgojitelji, ravnatelji predš-
kolskih ustanova i roditelji, a pokazalo se da su stavovi prema inkluziji
bili percipirani kao važna zapreka za inkluziju unutar dječjeg vrtića za
sve skupine ispitanika. Zanimljivo, baš svi roditelji su smatrali da su
stavovi edukatora iznimno važna barijera (100%) provođenju inkluziv-
nog obrazovanja. Stuber (1994) je provela istraživanje predškolskih spe-
cijalnih edukatora2 defektologa te ravnatelja dječjih vrtića i osnovnih
škola, osnovnoškolskih učitelja te odgojitelja u Kansasu. Ispitivali su se
stavovi sudionika prema inkluziji djece s teškoćama u razvoju u redovi-
te programe škola i dječjih vrtića. Rezultati su pokazali da su specijal-
ni edukatori imali pozitivnije stavove prema integraciji od odgojitelja
redovnog vrtića. Odgojitelji percipiraju prilagodbu nastavnih strategija
djeci s teškoćama u razvoju kao značajnu prepreku, jer svoja postoje-
ća znanja te postojeće materijalne preduvjete provođenja tih strategija
smatraju nedostatnim. U ispitivanju predškolskih djelatnika (odgoji-
telja, ravnatelja, te kombinirane radne uloge ravnatelj-odgojitelj) kori-
šteno je više instrumenata za ispitivanje spremnosti za inkluziju djece
u redovite odgojne skupine dječjeg vrtića (Eiserman, Shisler & Healey,
1995). 2 U Hrvatskoj, slični profili stručnjaka nazivaju se edukacijsko-rehabilita-
cijski stručnjaci ili defektolozi. Istraživanja stavova odgojitelja prema inkluziji u praksi
predškolskog odgoja i obrazovanja Rezultati su pokazali da sudionici svih grupa pokazuju umjereno
pozitivan stav prema inkluziji, i s aspekta djece s teškoćama u razvoju
i s aspekta ostale djece. U drugoj studiji (Gemmell-Crosby & Hanzlik,
1994) ispitani su stavovi odgojitelja prema inkluziji djece u dječjim vr-
tićima u SAD, težeći ispitati odnos između stavova odgojitelja prema
2
U Hrvatskoj, slični profili stručnjaka nazivaju se edukacijsko-rehabilita- 313 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. inkluziji i njihove percepcije vlastite kompetentnosti u zadovoljavanju
potreba djece u svojim odgojnim grupama, percepcije podrške njihovih
napora uloženih u inkluziju, njihovog obrazovanja, radnog iskustva, po-
laženja treninga. Pronađen je pozitivan odnos stavova prema inkluziji
sa autopercepcijom kompetentnosti, primljenom podrškom i zadovolj-
stvom obukom i obrazovanjem. Čang (Chung, 2000) je ispitivao stavove
prema kompetentnosti za inkluzivno obrazovanje, pokazavši da su mi-
šljenja odgojitelja o važnosti profesionalne kompetencije bila značajno
različita, ovisno o vrstama teškoća u razvoju, radnom odgojno-obrazov-
nom iskustvu vezanom uz inkluziju. Odgojitelji koji su bolje educirani
o određenoj vrsti teškoća u razvoju i koji su iskusniji u radu u uvjetima
inkluzije, imaju pozitivnije stavove prema inkluzivnom obrazovanju. Osim toga, odgojitelji su ukazali na potrebu za povećanjem praktičnih
vještina, i na razini znanja i na razini primjene u praksi. Čeng (Cheng,
2001) je ispitao kako tajvanski roditelji i odgojitelji percipiraju inkluzi-
ju predškolske djece s teškoćama u razvoju u redovni program dječjeg
vrtića. Ispitujući razlike u percepciji inkluzije, pokazalo se da i roditelji
djece s teškoćama u razvoju i roditelji djece bez teškoća imaju umjere-
no pozitivne stavove prema inkluziji. S druge strane, redoviti i posebno
educirani odgojitelji (za rad s djecom s teškoćama u razvoju) imali su
pozitivne stavove prema inkluziji i njenim prednostima za svu djecu,
dok se posebno educirani odgojitelji smatraju kompetentnijim za rad s
djecom u uvjetima inkluzije (Cheng, 2001). j
j
j
g
Arsenijević-Puhalo i Matošević (2007) ispitali su stavove prema in-
kluziji u Bosni i Hercegovini. Ispitanici (odgojiteljice) su u dvije trećine
slučajeva mišljenja da boravak djece s teškoćama u razvoju u predškol-
skim ustanovama zajedno sa djecom koja nemaju razvojnih oštećenja
pomaže njihovu adekvatnu socijalizaciju i kompenzaciju nedostatka u
psihofizičkom funkcioniranju. Inkluzija na predškolskoj razini uvodi
dijete u svijet komunikacije sa vršnjacima i pomaže da se razvije samo-
pouzdanje i povjerenje u druge. S druge strane, inkluzija ove dvije grupe
djece potiče empatiju i humano reagiranje kod djece bez razvojnih po-
teškoća. Istraživanja stavova odgojitelja prema inkluziji u praksi
predškolskog odgoja i obrazovanja Iako većina anketiranih u inkluziji vidi prednost za djecu, jed-
na četvrtina se ne može složiti sa tim. Pretjerani angažman odgojitelja
oko djece s teškoćama u razvoju i zanemarivanje ostale djece, nedosta-
tak profesionalne supervizije, te prevelik broj djece u redovnim grupa-
ma i otpor ostale djece su najčešći problemi koji se navode u realizaciji 314 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće inkluzije. Razlozi za zauzimanje stava prema neopravdanosti inkluzije
u predškolskim ustanovama su slični onima koji su navedeni kao pro-
blemi u realizaciji inkluzije. Skoro trećina odnosi se na brojnost grupa,
jedna petina na neorganiziranu superviziju, a po desetina na nestručan
kadar i nepripremljenost djece s teškoćama u razvoju za druženje s osta-
lom djecom. Prema mišljenju većine ispitanika, tehnička pripremljenost
vrtića za inkluziju je nezadovoljavajuća, jer postoje arhitektonske barije-
re, neprilagođeni ulazi, neadaptirani sanitarni čvorovi, skučen prostor
i stube. U jedinom dostupnom istraživanju o stavovima odgojitelj/ica
djece predškolske dobi prema inkluziji u Hrvatskoj, pokazale su se razli-
ke u stavovima prema inkluziji prije i nakon edukacije o inkluziji, u pet
skupina varijabli: razumijevanje filozofije odnosno kriterija inkluzije,
ublažavanje negativnih stavova prema inkluziji, razumijevanje utjecaja
stručnih i osobnih kapaciteta odgajatelja i roditelja kao sudionika proce-
sa na rad u uvjetima inkluzije, razumijevanje i prihvaćanje kriterija od-
gojno-obrazovnog rada u uvjetima inkluzije te razumijevanje važnosti
timskog rada (Kubelka i Sindik, 2009). Sažimajući nalaze svih navedenih istraživanja o stavovima od-
gojitelja prema inkluziji, izgleda da odgojitelji sumnjaju u svoje kom-
petencije za primjenu inkluzije u praksi, smatrajući da su nedovoljno
osposobljeni za zadovoljavanje potreba djece s teškoćama u razvoju. Osim toga, odgojitelji imaju umjereno pozitivne stavove prema inklu-
ziji, smatrajući da djeca bez teškoća u razvoju mogu „profitirati” od
djeteta koje je u inkluziji unutar redovite odgojne grupe dječjeg vrtića
(Peck, Carlson & Helmstetter, 1992). Evaluacija specifičnog programa edukacije
odgojitelja o inkluziji Utvrđivanje specifičnih stavova odgojitelja iznimno su relevan-
tni za primjenu inkluzije u praksi, jer su oni potencijalni ili aktivni
nositelji inkluzije u neposrednom odgojno-obrazovnom radu. S dru-
ge strane, sadržaj svakog pojedinog programa edukacije za odgojitelje
je drugačiji, ovisno o odgojno-obrazovnom sustavu određene države,
vrsti predškolskog programa i predškolskih ustanova. Da bi se znalo
koliki efekat postiže određen program edukacije, treba ga primjere-
no prethodno navedenim faktorima (specifičnost države, programa, 315 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. ustanova) i evaluirati. Primjer jednog takvog pokušaja evaluacije, koji
je djelomično i bio polazište za konstrukciju upitnika u ovom radu,
dao je Čeng (Cheng, 2001). On je osmislio upitnike za roditelje i odgo-
jitelje prilagođene tajvanskom odgojno-obrazovnom sustavu, izdvo-
jivši šest dimenzija stavova odgojitelja: (1) Percepcija prednosti (od
strane odgojitelja) za djecu educiranu u inkluzivnom dječjem vrtiću;
(2) Odgojiteljska percepcija vlastite kompetentnosti za rad s djecom
u inkluzivnom dječjem vrtiću; (3) Odgojiteljska dobrovoljnost rada u
inkluzivnom dječjem vrtiću; (4) Odgojiteljska percepcija dostupnih re-
sursa za podršku inkluziji u postojećem redovitom programu dječjeg
vrtića; (5) Odgojiteljska percepcija vlastite vještine podučavanja u smi-
slu stimulacije pravilnog razvoja ostale djece u dječjim vrtićima (osim
djeteta s teškoćama u razvoju); (6) Odgojiteljska percepcija vlastite
vještine podučavanja djece s teškoćama u razvoju u dječjim vrtićima. Kubelka i Sindik (2009) su osmislili upitnik koji je pokrivao tematska
područja edukacije o inkluziji, koristeći teorijske postavke kurikulu-
ma za inkluziju (Daniels & Stafford, 2003), sa sadržajima: stjecanje
teoretskog znanja o kategorijama i obilježjima teškoća u razvoju; prak-
tičnih vježbi uživljavanja u iskustva nekih dječjih teškoća u razvoju
(poput disleksije, slabovidnosti, nagluhosti, motoričkih smetnji, itd.);
stjecanje praktičnih znanja o diferenciranim instrukcijama ovisno o
individualnim potrebama djeteta (načela individualnog plana rada
sa svakim djetetom); razmještaj prostorije i promjene potrebne kako
bi se podržao razvoj djeteta i učenje; znanja o učinkovitim odgojno-
obrazovnim strategijama koje stimuliraju djecu na suradničko učenje;
suradnja i razvijanje partnerstva s roditeljima kako bi se osigurale bo-
lje mogućnosti za obrazovanje i brigu o djeci. Sadržaj edukacije je ci-
ljano primjeren specifičnim područjima praktičnih potreba primje-
rene edukacije o inkluziji u uvjetima hrvatskog obrazovnog sustava,
usklađen s Državnim pedagoškim standardom predškolskog odgoja
i naobrazbe (2008) te teorijskim postavkama kurikuluma za inkluzi-
ju (Daniels & Stafford, 2003). Evaluacija specifičnog programa edukacije
odgojitelja o inkluziji Prema sadržaju edukacije, određeni su
potencijalno pogodni indikatori kvalitetnog rada u uvjetima inkluzije
u hrvatskom predškolskom odgojno-obrazovnom sustavu, svedeni na
stavove odgojitelja, koji su bili grupirani u sadržajno povezane temat-
ske cjeline: razumijevanje filozofije i kriterija rada u uvjetima inkluzi- 316 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće kapaciteti sudionika inkluzije (Kubelka i Sindik, 2009). Na temelju
saznanja o tim stavovima, moglo bi se ne samo modificirati postojeći
program edukacije, već ga i prilagoditi svakoj pojedinoj skupini pola-
znika edukacije. Dakle, temeljni cilj istraživanja bio je konstruirati mjerni instru-
ment za mjerenje stavova odgojitelja praktičara prema inkluziji od če-
stica njegove preliminarne verzije. U skladu s temeljnim ciljem, for-
mulirali smo i probleme (potciljeve) istraživanja: 1. Utvrditi latentne dimenzije u osnovi stavova prema inkluziji. 2. Utvrditi međusobnu povezanost dimenzija stavova prema inkluziji Pretpostavili smo da postoje latentne dimenzije na temelju ko-
jih bismo mogli reducirati prostor manifestnih varijabli koje pokriva-
ju područja stavova odgojitelja praktičara prema inkluziji. Nismo mo-
gli unaprijed pretpostaviti koliki će biti broj tih dimenzija. Međutim,
predvidjeli smo da će jedna od faktorskih solucija, koja daje pouzdane i
interpretabilne dimenzije, pokrivati barem nekolicinu od navedenih po-
dručja edukacije o inkluziji. Premda već na temelju različitosti područja
stavova prema inkluziji nismo mogli očekivati visoke korelacije između
pojedinih dimenzija stavova odgojitelja prema inkluziji, mogli smo pret-
postaviti da će određen broj dimenzija biti statistički značajno povezan. 3 Zbog lakše razumljivosti odgojiteljima-praktičarima, u upitniku je ciljano
korišten neslužbeni a u praksi vrlo rašireni termin „posebne potrebe” umjesto
djece s teškoćama u razvoju. Naravno, u samoj je edukaciji naglašena razlika ovog
termina od zakonskog „djeca s teškoćama u razvoju”. Sudionici U istraživanju je sudjelovalo 159 odgojiteljica iz redovitih dječjih vr-
tića: 75 odgojiteljica iz četiri dječja vrtića, tri u Zagrebu: 14, 35 i 26 odgo-
jitelja i jednog u Osijeku (84). Prosječna dob odgojiteljica bila je 40 godina,
a dob im je varirala od 24 do 58 godina (35 odgojiteljica do 30 godina, 55
do 40 godina, 45 do 50 godina te 24 odgojiteljice preko 50 godina). Radni
staž je pak varirao od dvije do 34 godine. U odgojnim grupama u kojima
odgojiteljice rade bilo je od niti jednog djeteta (kod 123 odgojiteljice) do
maksimalno troje djece s teškoćama u razvoju, a većina odgojiteljica (132)
je imala tijekom radnog staža iskustva u radu s djecom s teškoćama u ra-
zvoju, tijekom barem jednogodišnjeg razdoblja. Međutim, sve odgojiteljice
su radile s djecom s teškoćama u razvoju, dakle s privremenim i potenci-
jalnim posebnim potrebama (Programsko usmjerenje odgoja i obrazova- 317 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. nja predškolske djece, 1991), barem godinu dana. Sve odgojiteljice završile
su odgojiteljsku školu: čak 137 višu, a preostale (22) srednju odgojiteljsku
školu (u ovom slučaju, riječ je o odgojiteljicama s više od 25 godina radnog
staža). Dakle, usprkos određenim odstupanjima, uzorak smo u pogledu
relevantnih faktora za ovo istraživanje mogli smatrati prilično homoge-
nim po relevantnim karakteristikama (podjednaka razina obrazovanja,
dovoljno radno iskustvo, iskustvo u radu s djecom s teškoćama u razvoju). Prije provođenja istraživanja, odgojiteljice su dale informirani pristanak
za sudjelovanje u istraživanju. Metode obrade podataka Izračunate su mjere deskriptivne statistike za sve čestice upit-
nika, a nakon faktorske analize i za dimenzije definirane kao jedno-
stavne linearne kombinacije čestica koje definiraju pojedini faktor. Da
bi se utvrdila struktura dimenzija koja je u osnovi različitih varijabli
stavova prema inkluziji, proveden je postupak komponentne analize:
broj značajnih glavnih komponenata određen je GK (Guttman-Kai-
ser) kriterijem, a potom je izračunata matrica korelacija sa značajnim
glavnim osovinama. Kao metoda ekstrakcije primijenjena je metoda
glavnih komponenata, a potom Varimax rotacija na normalizirana
faktorska opterećenja, kako opisuje Mejovšek (2003). Nakon svake od
iteracija komponentne analize, Cronbach’s α koeficijentom izračunali
smo pouzdanost pojedinih faktora, tj. dobivenih dimenzija upitnika,
ali i pouzdanost upitnika bez svake od čestica, čije su visine interpre-
tirane prema kriterijima koje daje Nunnally (1978). Na temelju GK-
kriterija, te interpretabilnosti faktora, došli smo do konačne faktorske
solucije. Na kraju, izračunali smo i interkorelacije između pojedinih
dimenzija upitnika. Premda se omjer broja slučajeva i broja varijabli
u ovom istraživanju doima premali za provođenje faktorske analize,
napominjemo da Zhao (2009) navodi da je čak omjer od 3:1 (Cattell,
1978, from Zhao, 2009) te čak 2:1 (Kline, 1979, from Zhao, 2009) za-
dovoljavajući za provedbu faktorske analize. Mjerni instrument Prije edukacije o inkluziji, primijenjena je preliminarna (pilot)
verzija upitnika „Stavovi stručnjaka prema inkluziji djece s teškoća-
ma u razvoju”, koji su isključivo za praktične namjene sastavili autori
rada3. Ova inicijalna verzija Upitnika, kojem nisu bile provjeravane
metrijske karakteristike (te ga nismo mogli smatrati mjernim instru-
mentom), sastojala se od 86 tvrdnji (Kubelka i Sindik, 2009), na kojoj
su sudionici na skali Likertova tipa od pet stupnjeva, imali zadatak
izraziti stupanj slaganja sa sadržajem tvrdnje na ljestvici u rasponu od
„1” (uopće se ne slažem) do „5” (u potpunosti se slažem). Već u prelimi-
narnoj verziji Upitnika, na temelju diskriminativnosti pojedinih česti-
ca (indeks relativnog varijabiliteta), reducirali smo broj čestica za ver-
ziju korištenu u ovom istraživanju na 78 čestica. Jedna jedina čestica
(r.b. 25) koja je u negativnoj korelaciji s dimenzijom koju definira, bila
je rekodirana u jednostavnoj linearnoj kombinaciji. U ovom smo istra-
živanju provjerili metrijske karakteristike te radne verzije Upitnika
s reduciranim brojem od 78 čestica, utvrdivši njegovu pouzdanost i
konstruktnu valjanost. Sudionici su Upitnike ispunjavali anonimno,
uz naglašenu isključivo znanstvenu svrhu istraživanja. 318 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće Latentne dimenzije osnovnih stavova
odgojiteljica prema inkluziji U svrhu provjere konstruktne valjanosti, inicijalno smo provje-
rili pogodnost rezultata za faktorizaciju Keiser-Meyer-Olkinovom
mjerom adekvatnosti uzorka koja je pokazala nisku, ali zadovolja-
vajuću vrijednost (0,58). S druge strane, Bartlettov test sfericiteta
(χ2=5684,02; p<0,001) ukazuje na podatak da korelacijska matrica bit-
no odstupa od matrice identiteta, dakle podaci ne odstupaju od uzor-
ka iz multivarijatne normalne populacije. Dobiveno je sedam faktora,
koji zajedno tumače ukupno 41,74 % zajedničke varijance, koje smo 319 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. imenovali na temelju sadržaja područja edukacije o inkluziji (Udru-
ge „Korak po korak”). Ukupna količina objašnjene varijance bila je
prilično mala, ali nipošto neuobičajena za istraživanja u društvenim
znanostima. Najvjerojatniji razlog relativno male količine objašnjene
varijance, odnosno dobivene latentne strukture upitnika mogao bi biti
relativno nehomogen konstrukt stavova odgojiteljica prema inkluziji
djece u uvjetima redovitih dječjih vrtića. Naime, stavovi prema inklu-
ziji su i teorijski (a ne samo metrijski) dosta širok i relativno neho-
mogen konstrukt, dok s druge strane očigledno postoji heterogenost
stavova prema različitim aspektima inkluzije među odgojiteljima. Pokazalo se, dakle, da postoji sedam latentnih dimenzija koje de-
finiraju prostor stavova odgojiteljica prema inkluziji djece u uvjetima
redovitih (nespecijaliziranih) dječjih vrtića: kompetencija odraslih,
otvorenost dječjeg vrtića, organizacijske prilagodbe, predrasude pre-
ma inkluziji, filozofija inkluzije, kriteriji praćenja inkluzije i osobne
i stručne kompetencije. Rezultati faktorske analize čestica upitnika
Stavovi stručnjaka prema inkluziji djece s teškoćama u razvoju prika-
zani su u tablicama 1-7. Dimenzija kompetencija odraslih (Tablica 1) opisuje relaciju
uspješne inkluzije i pravilnog odnosa odraslih prema djetetu koje je u
procesu inkluzije (primjereno ponašanje roditelja, stručnih suradnika,
odgojiteljica, ravnatelja dječjeg vrtića u smislu stimuliranja uspješne
inkluzije). Prosječna aritmetička sredina za dimenziju kompetencija
odraslih (aritmetička sredina podijeljena brojem čestica koje defini-
raju dimenziju, tj. 12) iznosila je 3,93, dok je prosječno standardno
raspršenje iznosilo 0,48. U preliminarnoj verziji dimenziju je repre-
zentiralo 15 čestica. Latentne dimenzije osnovnih stavova
odgojiteljica prema inkluziji 320 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće inkluziji djece s teškoćama u razvoju u dječje vrtiće
Tablica 1 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor
kompetencija odraslih)
Faktor kompetencija odraslih
Korelacije
varijabli s
faktorom
Prosječna
interkorelacija
s drugim
česticama
Cronbach’s α
bez čestice
M
σ
Uspješnost integracije djeteta s TUR ovisi o
ličnosti stručnog suradnika koji radi s djetetom
0,60
0,44
0,73
3,83
0,94
Uspješnost integracije djeteta s TUR ovisi o
stupnju educiranosti roditelja
0,54
0,50
0,73
3,90
,94
Korištenje vizualnih uputa korisno je kad dijete
usvaja rutine
0,54
0,45
0,74
4,20
,62
Uspješnost integracije djeteta s TUR ovisi o
stupnju afiniteta ravnatelja za problematiku
djece s TUR
0,53
0,41
0,74
3,18
1,11
Integracija je potrebna za svu djecu
0,50
0,27
0,76
4,42
0,79
Uspješnost integracije djeteta s TUR ovisi o
ličnosti (oba) roditelja
0,46
0,42
0,74
3,86
1,01
Cilj inkluzije je postići da dijete postigne sve
ono što bi prema razvojnim mogućnostima
moglo ostvariti (prema dobnim mogućnostima
djeteta)
0,44
0,37
0,74
3,56
1,12
Uspješnost integracije djeteta s TUR ovisi o
kvaliteti suradnje s roditeljima
0,42
0,39
0,74
4,29
0,84
Uspješnost integracije djeteta s TUR ovisi o
stupnju afiniteta pojedinog stručnog suradnika
za problematiku djece s TUR
0,42
0,40
0,74
3,83
0,78
Inkluzija kao sustav ima polazište u „jakim”
stranama svakog djeteta
0,41
0,37
0,74
4,00
0,83
Individualizirani pristup je najdjelotvorniji
način rada s djecom s TUR integriranom u
redovite odgojne grupe vrtića
0,39
0,35
0,75
4,21
0,94
Poznavanje strategija smislenog učenja vrlo je
važno za proces inkluzije
0,34
0,40
0,74
3,79
0,80
Pokazatelj
Karakteristični
korijen
Postotak
objašnjene
varijance (%)
Pouzdanost
ljestvice
M dimenzije
σ dimenzije
3,75
7,22
0,76
47,12 5,71 Faktor kompetencija odraslih Pokazatelj 321 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. Tablica 2 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor
predrasude prema inkluziji)
Faktor predrasude prema inkluziji
Korelacije
varijabli s
faktorom
Prosječna
interkorelacija
s drugim
česticama
Cronbach’s α
bez čestice
M
σ
Za integraciju je potrebno zadovoljiti puno više
uvjeta nego za proces inkluzije
0,67
0,33
0,58
2,99
1,05
Integracija je nadređen pojam inkluziji
0,57
0,23
0,60
3,33
0,92
Ako je dijete „vizualni” tip, treba ga najprije
učiti usvajanju novih pojmova i vještina putem
slušnog kanala
0,54
0,41
0,55
2,87
1,32
Socijalizacija i inkluzija djeteta s TUR je jedan te
isti pojam
0,54
0,44
0,55
2,88
1,08
Za rad u inkluziji nije presudna kvaliteta
odgojno-obrazovne prakse u vrtiću
0,46
0,26
0,59
2,38
1,08
Integracija stavlja odgovornost na dijete da
udovolji standardima
0,42
0,32
0,58
2,69
1,02
Pojmovi „posebne potrebe” i „teškoće u razvoju”
imaju isto značenje
0,42
0,26
0,60
2,52
1,36
Proces integracije djeteta s TUR nastoji
ujednačiti dijete s TUR s „prosječnom” djecom u
odgojnoj grupi vrtića
0,36
0,40
0,56
3,22
1,12
Integracija se nužno provodi pomoću direktnog
rada s djetetom u odgojnoj grupi, pri čemu se
nalaze načini poticanja djetetova razvoja
0,34
0,24
0,60
3,89
0,87
Pokazatelj
Karakteristični
korijen
Postotak
objašnjene
varijance (%)
Pouzdanost
ljestvice
M dimenzije
σ dimenzije
3,35
6,64
0,61
26,96
4,94
Dimenzija predrasude prema inkluziji (Tablica 2) opisuje pojedno-
stavljene stavove, vjerojatno ponajviše utemeljene na površnom pristupu
edukaciji o inkluziji, čega je vjerojatna posljedica nedovoljno učinkovit pri-
stup inkluziji djeteta u praktičnom odgojno-obrazovnom radu. Prosječna
aritmetička sredina za dimenziju predrasude prema inkluziji (aritme-
tička sredina podijeljena brojem od 9 čestica koje definiraju dimenziju)
iznosila je 2,99, dok je prosječno standardno raspršenje iznosilo 0,58. U
l
d
l Pokazatelj Dimenzija predrasude prema inkluziji (Tablica 2) opisuje pojedno-
stavljene stavove, vjerojatno ponajviše utemeljene na površnom pristupu
edukaciji o inkluziji, čega je vjerojatna posljedica nedovoljno učinkovit pri-
stup inkluziji djeteta u praktičnom odgojno-obrazovnom radu. Prosječna
aritmetička sredina za dimenziju predrasude prema inkluziji (aritme-
tička sredina podijeljena brojem od 9 čestica koje definiraju dimenziju)
iznosila je 2,99, dok je prosječno standardno raspršenje iznosilo 0,58. U
preliminarnoj verziji dimenziju je reprezentiralo 15 čestica. 322 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće Tablica 3 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor otvorenost
dječjeg vrtića) dječjeg vrtića)
Faktor otvorenost dječjeg vrtića
Korelacije varijabli
s faktorom
Prosječna
interkorelacija s
drugim česticama
Cronbach’s α bez
čestice
M
σ
Stručni suradnici u dječjim vrtićima djelotvorno
rade s djecom s TUR
0,66
0,58
0,63
2,96
1,13
Postojeći uvjeti u dječjim vrtićima omogućavaju
djelotvornu integraciju djeteta s TUR
0,60
0,45
0,66
2,30
1,06
Djecu s teškoćama u razvoju treba uključiti u
redovite odgojne grupe vrtića u trajanju od 10
sati (cjelodnevni program)
0,55
0,43
0,67
2,49
1,21
Stručnjaci iz specijaliziranih vanjskih institucija
(bolnice, SUVAG i sl.) daju konstruktivne upute
vezane uz integraciju djece s TUR
0,50
0,37
0,69
2,94
1,28
Odgojitelji predškolske djece vole raditi s djecom
s TUR
0,49
0,38
0,68
2,72
0,92
Dijete s TUR treba imati prioritet pri upisu u
dječji vrtić
0,46
0,40
0,68
3,00
1,20
Inkluzija redovito rezultira uspješnom
socijalizacijom djeteta s TUR
0,46
0,37
0,68
3,43
0,88
Rad u uvjetima inkluzije ne razlikuje se od rada
u uvjetima integracije
0,42
0,20
0,71
2,97
0,82
Pokazatelj
Karakteristični korijen
Postotak objašnjene
varijance (%)
Pouzdanost ljestvice
M dimenzije
σ dimenzije
3,32
6,45
0,71
22,74
4,90
Dimenzija otvorenost dječjeg vrtića (Tablica 3) opisuje stav da je
inkluzija općenito poželjna u dječjim vrtićima, i na formalnoj razini
(poželjnost uključivanja djece s posebnim potrebama u programe vrti-
ća), i na praktičnoj razini (vjerovanje u uspjeh inkluzije te u kreativnu
interakciju stručnjaka iz dječjeg vrtića i izvan njega u pogledu uspješ- Pokazatelj U preliminarnoj verziji
dimenziju je reprezentiralo 9 čestica Tablica 4 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor osobne i
stručne kompetencije) Tablica 4 – Faktorska analiza upitnika Stavovi stručnjaka prema
nkluziji djece s teškoćama u razvoju (varimax rotacija – faktor osobne i
tručne kompetencije) Faktor osobne i stručne kompetencije
Korelacije
varijabli s
faktorom
Prosječna
interkorelacija s
drugim česticama
Cronbach’s α bez
čestice
M
σ
Uspješnost integracije djeteta s TUR ovisi o
stupnju afiniteta pojedinih odgojitelja za rad s
djetetom s TUR
0,71
0,57
0,58
3,92
0,93
Uspješnost integracije djeteta s TUR ovisi
o iskustvu i refleksivnosti odgojitelja
(senzibiliziranosti za potrebe djeteta)
0,66
0,57
0,57
4,21
0,74
Uspješnost integracije djeteta s TUR ovisi o
ličnosti (oba) odgojitelja
0,64
0,49
0,59
4,09
0,92
Vrtići u našem gradu imaju nedovoljan broj
stručnjaka za rad s djecom s TUR
0,54
0,30
0,68
4,25
1,11
Za kvalitetu rada u inkluziji presudan je stav
odgojitelja prema djetetu
0,39
0,32
0,66
3,97
0,98
Pokazatelj
Karakteristični
korijen
Postotak
objašnjene
varijance (%)
Pouzdanost
ljestvice
M dimenzije
σ dimenzije
3,30
6,34
0,67
20,41
3,10 Pokazatelj Dimenzija otvorenost dječjeg vrtića (Tablica 3) opisuje stav da je
inkluzija općenito poželjna u dječjim vrtićima, i na formalnoj razini
(poželjnost uključivanja djece s posebnim potrebama u programe vrti-
ća), i na praktičnoj razini (vjerovanje u uspjeh inkluzije te u kreativnu
interakciju stručnjaka iz dječjeg vrtića i izvan njega u pogledu uspješ-
ne inkluzije djeteta s posebnim potrebama u dječjem vrtiću). Prosječna 323 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. aritmetička sredina za dimenziju otvorenost dječjeg vrtića (aritme-
tička sredina podijeljena brojem od 8 čestica koje definiraju dimenziju)
iznosila je 2,84, dok je prosječno standardno raspršenje iznosilo 0,61. U
preliminarnoj verziji dimenziju je reprezentiralo 12 čestica. Tablica 4 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor osobne i
stručne kompetencije) aritmetička sredina za dimenziju otvorenost dječjeg vrtića (aritme-
tička sredina podijeljena brojem od 8 čestica koje definiraju dimenziju)
iznosila je 2,84, dok je prosječno standardno raspršenje iznosilo 0,61. U
preliminarnoj verziji dimenziju je reprezentiralo 12 čestica. preliminarnoj verziji dimenziju je reprezentiralo 12 čestica. Tablica 4 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor osobne i
stručne kompetencije)
Faktor osobne i stručne kompetencije
Korelacije
varijabli s
faktorom
Prosječna
interkorelacija s
drugim česticama
Cronbach’s α bez
čestice
M
σ
Uspješnost integracije djeteta s TUR ovisi o
stupnju afiniteta pojedinih odgojitelja za rad s
djetetom s TUR
0,71
0,57
0,58
3,92
0,93
Uspješnost integracije djeteta s TUR ovisi
o iskustvu i refleksivnosti odgojitelja
(senzibiliziranosti za potrebe djeteta)
0,66
0,57
0,57
4,21
0,74
Uspješnost integracije djeteta s TUR ovisi o
ličnosti (oba) odgojitelja
0,64
0,49
0,59
4,09
0,92
Vrtići u našem gradu imaju nedovoljan broj
stručnjaka za rad s djecom s TUR
0,54
0,30
0,68
4,25
1,11
Za kvalitetu rada u inkluziji presudan je stav
odgojitelja prema djetetu
0,39
0,32
0,66
3,97
0,98
Pokazatelj
Karakteristični
korijen
Postotak
objašnjene
varijance (%)
Pouzdanost
ljestvice
M dimenzije
σ dimenzije
3,30
6,34
0,67
20,41
3,10
Dimenzija osobne i stručne kompetencije (Tablica 4) opisuje stav
da uspješnost inkluzije djece s posebnim potrebama u programe vrti-
ća ovisi o odgojiteljima kao osobama, njihovom osobnom angažma-
nu i stavu prema djetetu. Prosječna aritmetička sredina za dimenziju
osobne i stručne kompetencije (aritmetička sredina podijeljena
brojem od 5 čestica koje definiraju dimenziju) iznosila je 4,08, dok je
prosječno standardno raspršenje iznosilo 0,62. Pokazatelj Dimenzija osobne i stručne kompetencije (Tablica 4) opisuje stav
da uspješnost inkluzije djece s posebnim potrebama u programe vrti-
ća ovisi o odgojiteljima kao osobama, njihovom osobnom angažma-
nu i stavu prema djetetu. Prosječna aritmetička sredina za dimenziju
osobne i stručne kompetencije (aritmetička sredina podijeljena
brojem od 5 čestica koje definiraju dimenziju) iznosila je 4,08, dok je
prosječno standardno raspršenje iznosilo 0,62. U preliminarnoj verziji
dimenziju je reprezentiralo 9 čestica. 324 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće inkluziji djece s teškoćama u razvoju u dječje vrtiće
Tablica 5 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor
organizacijske prilagodbe)
Faktor organizacijske prilagodbe
Korelacije
varijabli s
faktorom
Prosječna
interkorelacija s
drugim česticama
Cronbach’s α bez
čestice
M
σ
Rad u uvjetima inkluzije pretpostavlja
uključivanje stručnog tima u neposredan
odgojno-obrazovni rad
0,69
0,42
0,41
4,70
0,56
Rad u uvjetima inkluzije pretpostavlja dobru
organizaciju sustava podrške u vrtiću
0,65
0,46
0,39
4,57
0,64
Ako tim stručni suradnik –odgojitelj- roditelj ne
rade zajedno na IOOP, teže će se realizirati rad u
uvjetima inkluzije
0,58
0,41
0,39
4,52
0,74
Rad u uvjetima inkluzije manje je uspješan ako
odgojitelji rade u uvjetima koji nisu u skladu s
novim pedagoškim standardima (prevelik broj
djece)
0,54
0,34
0,42
4,60
0,76
Za uspješnu inkluziju potrebni su nam jasno
razrađeni modeli uključivanja svakog pojedinog
djeteta s TUR u redovite odgojne skupine vrtića
0,47
0,23
0,47
4,40
0,70
Jedan od razloga što više djece s TUR nije
uključeno u institucionalni oblik predškolskog
odgoja i obrazovanja je taj što predškolski odgoj i
obrazovanje nisu obavezni
-0,34
0,10
0,56
2,99
1,02
Pokazatelj
Karakteristični
korijen
Postotak
objašnjene
varijance (%)
Pouzdanost
ljestvice
M dimenzije
σ dimenzije
2,93
5,63
0,50
25,81
2,57
Dimenzija organizacijske prilagodbe (Tablica 5) opisuje stav da je
za uspješnost inkluzije djece s posebnim potrebama u programe vrtića
iznimno bitna prilagodba organizacije rada u dječjem vrtiću, ali i u
sustavu odgoja i obrazovanja općenito. Prosječna aritmetička sredina
za dimenziju organizacijske prilagodbe (aritmetička sredina podi-
jeljena brojem od 6 čestica koje definiraju dimenziju) iznosila je 4,30,
prosječno standardno raspršenje iznosilo 0,43. U preliminarnoj verziji
dimenziju je reprezentiralo 8 čestica. Pokazatelj Dimenzija organizacijske prilagodbe (Tablica 5) opisuje stav da je
za uspješnost inkluzije djece s posebnim potrebama u programe vrtića
iznimno bitna prilagodba organizacije rada u dječjem vrtiću, ali i u
sustavu odgoja i obrazovanja općenito. Prosječna aritmetička sredina
za dimenziju organizacijske prilagodbe (aritmetička sredina podi-
jeljena brojem od 6 čestica koje definiraju dimenziju) iznosila je 4,30,
prosječno standardno raspršenje iznosilo 0,43. U preliminarnoj verziji
dimenziju je reprezentiralo 8 čestica. 325 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. Tablica 6 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor
filozofija inkluzije)
Faktor filozofija inkluzije
Korelacije
varijabli s
faktorom
Prosječna
interkorelacija
s drugim
česticama
Cronbach’s α
bez čestice
M
σ
Inkluzija podrazumijeva uključenost djece
s posebnim potrebama, od najranijih dana,
ne samo u odgojno-obrazovni sustav, već i u
svakodnevnu društvenu i životnu stvarnost
0,67
0,53
0,45
4,27 0,82
Inkluzija je proces učenja i odgajanja djece s
posebnim potrebama zajedno s djecom koja
nemaju takvih potreba
0,55
0,38
0,52
4,25
0,78
Integracija djeteta s teškoćom u praksi najčešće
znači da ono dio vremena boravi u igri s djecom
u redovitim odgojnim grupama vrtića, dok je
ostatak vremena uključeno u rehabilitacijski
program
0,53
0,26
0,57
3,91 1,00
Inkluzija stavlja odgovornost na predškolski
sustav da udovolji potrebama sve djece
0,52
0,37
0,52
3,82 0,92
Inkluzija zahtijeva puno veću odgovornost
stručnih djelatnika vrtića prema djetetu i
roditelju djeteta s TUR
0,40
0,32
0,54
4,22
0,72
Odgojitelji u pravilu nisu odgovarajuće
osposobljeni za rad s djecom s TUR
0,39
0,17
0,63
3,92
1,11
Pokazatelj
Karakteristični
korijen
Postotak
objašnjene
varijance (%)
Pouzdanost
ljestvice
M dimenzije
σ dimenzije
Dimenzija filozofija inkluzije (Tablica 6) objedinjuje ispravna zna-
nja ili stavove prema prirodi i preduvjetima uspješne inkluzije. Pro-
sječna aritmetička sredina za dimenziju filozofija inkluzije (aritme-
tička sredina podijeljena brojem od 6 čestica koje definiraju dimenziju)
iznosila je 4,06, dok je prosječno standardno raspršenje iznosilo 0,51. U preliminarnoj verziji dimenziju je reprezentiralo 9 čestica. Faktor filozofija inkluzije 4 Prividno se u Upitniku na nekim mjestima pojmovi integracije i inkluzije
koriste gotovo kao sinonimi; međutim, cilj ovakvih formulacija daje informaciju
edukatorima o eventualnoj potrebi dodatnog razjašnjavanja razlika između ova
dva pojma. Pokazatelj Dimenzija filozofija inkluzije (Tablica 6) objedinjuje ispravna zna-
nja ili stavove prema prirodi i preduvjetima uspješne inkluzije. Pro-
sječna aritmetička sredina za dimenziju filozofija inkluzije (aritme-
tička sredina podijeljena brojem od 6 čestica koje definiraju dimenziju)
iznosila je 4,06, dok je prosječno standardno raspršenje iznosilo 0,51. U preliminarnoj verziji dimenziju je reprezentiralo 9 čestica. 326 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće inkluziji djece s teškoćama u razvoju u dječje vrtiće
Tablica 7 – Faktorska analiza upitnika Stavovi stručnjaka prema
inkluziji djece s teškoćama u razvoju (varimax rotacija – faktor
filozofija inkluzije)
Faktor kriteriji praćenja inkluzije4
Korelacije
varijabli s
faktorom
Prosječna
interkorelacija
s drugim
česticama
Cronbach’s α
bez čestice
M
Σ
Dijagnoze koju daju stručnjaci iz
specijaliziranih vanjskih institucija u vezi sa
djecom s TUR su pogrešne
0,56
0,31
0,44
2,24
0,92
Individualizirani programi ključni su za
osiguravanje procesa integracije, ali ne i za
inkluziju
0,52
0,33
0,43
3,04
0,87
Uspješnost integracije djeteta s TUR ovisi o
dobi djeteta
0,50
0,28
0,46
3,05
1,10
Mišljenja koja daju stručnjaci iz dječjih
vrtića u vezi s djecom s TUR su pogrešna
0,46
0,21
0,50
2,43
1,01
Integracija podrazumijeva da svako dijete
ako može polazi dječji vrtić
0,44
0,28
0,46
3,99
0,96
Inkluzija u Hrvatskoj nije prihvaćena kao
jedini način uključivanja djece s posebnim
potrebama u vrtić
0,41
0,18
0,50
3,14
0,68
Pokazatelj
Karakteristični
korijen
Postotak
objašnjene
varijance (%)
Pouzdanost
ljestvice
M dimenzije
σ dimenzije
2,28
4,39
0,51
17,90
9,10
Konačno, dimenzija kriteriji praćenja inkluzije (Tablica 7) obje-
dinjuje stavove prema faktorima koji su bitni za praćenje procesa in-
kluzije (postojanje individualiziranih programa, mišljenja stručnjaka
iz specijaliziranih institucija, dob djeteta). Prosječna aritmetička sre-
dina za dimenziju kriteriji praćenja inkluzije (aritmetička sredina
podijeljena brojem od 6 čestica koje definiraju dimenziju) iznosila je
2,99, dok je prosječno standardno raspršenje iznosilo 0,51. U prelimi-
narnoj verziji dimenziju je reprezentiralo 10 čestica. Pokazatelj Konačno, dimenzija kriteriji praćenja inkluzije (Tablica 7) obje-
dinjuje stavove prema faktorima koji su bitni za praćenje procesa in-
kluzije (postojanje individualiziranih programa, mišljenja stručnjaka
iz specijaliziranih institucija, dob djeteta). Prosječna aritmetička sre-
dina za dimenziju kriteriji praćenja inkluzije (aritmetička sredina
podijeljena brojem od 6 čestica koje definiraju dimenziju) iznosila je
2,99, dok je prosječno standardno raspršenje iznosilo 0,51. U prelimi-
narnoj verziji dimenziju je reprezentiralo 10 čestica. 327 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. Dimenzije pokazuju nisku ali granično zadovoljavajuću pouzda-
nost, koja varira (izražena Cronbachovim α-koeficijentom) od 0,50 do
0,76. Pouzdanost pojedinih dimenzija stavova prema inkluziji je za tri
skale vrlo niska (tj. loša): organizacijska prilagodba, kriteriji praćenja
inkluzije, filozofija inkluzije. U dimenzijama kompetencija odraslih i
otvorenost dječjeg vrtića, pouzdanost je osrednje zadovoljavajuća (pri-
hvatljiva), dok je za preostale dimenzije pouzdanost niska (upitna). Međusobna povezanost dimenzija stavova prema inkluziji U Tablici 8 vidljivo je da korelacije među dimenzijama upitnika
variraju u rasponu od -0,11 do 0,43. Od ukupno 21 korelacije, samo
ih 8 nije statistički značajno. Dimenzije stavova prema inkluziji (koje
su statistički značajne) su pretežno međusobno nisko, ali statistički
značajno i pozitivno povezane. Otvorenost dječjeg vrtića te osobne i
stručne kompetencije najslabije su povezane s ostalim dimenzijama. Dimenzija kompetencija odraslih najbolje je povezana s ostalim di-
menzijama. S kronološkom dobi sudionica statistički značajno nisu
povezane dimenzije kompetencija odraslih, otvorenost dječjeg vrtića,
organizacijske prilagodbe. Statistički značajno i pozitivno su s krono-
loškom dobi povezane dimenzije predrasude prema inkluziji, filozofi-
ja inkluzije, kriteriji praćenja inkluzije. Jedina negativna a statistički
značajna povezanost pronađena je kod dimenzije osobne i stručne
kompetencije, s kronološkom dobi. 328 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće Tablica 8 – Korelacije između dimenzija Upitnika o stavovima
stručnjaka prema inkluziji djece s teškoćama u razvoju
Varijable
Kompetencija
odraslih
predrasude
prema
inkluziji
otvorenost
dječjeg
vrtića
osobne i
stručne
kompetencije
organizacijske
prilagodbe
filozofija
inkluzije
kriteriji
praćenja
inkluzije
kronološka dob
0,08
0,16*
-0,01
-0,18*
0,13
0,27**
0,23**
kompetencija
odraslih
1
0,33**
0,32**
0,43**
0,21**
0,38**
0,25**
predrasude
prema inkluziji
1
0,27**
0,02
-0,11
0,18*
0,27**
otvorenost
dječjeg vrtića
1
0,15
-0,02
0,14
0,05
osobne i
stručne
kompetencije
1
0,14
0,24**
0,07
organizacijske
prilagodbe
1
0,22**
0,01
filozofija
inkluzije
1
0,22**
kriteriji
praćenja
inkluzije
10
Legenda: **statistička značajnost p<0,01; *statistička značajnost p<0,05 Tablica 8 – Korelacije između dimenzija Upitnika o stavovima
stručnjaka prema inkluziji djece s teškoćama u razvoju 10 nda: **statistička značajnost p<0,01; *statistička značajnost p<0,05 Premda su ove dimenzije dijelom statistički značajno (najčešće
pozitivno) povezane, korelacije među njima su niske. Međutim, ovakvi
rezultati mogu biti očekivani iz razloga što spoznaje o različitim as-
pektima problematike inkluzije obuhvaćaju istovremeno i međusobno
povezana, ali ipak različita područja. Osobito je zanimljiv podatak da
predrasude prema inkluziji (kao jedina dimenzija nepoželjnih stavova
prema inkluziji) pokazuju pozitivnu povezanost čak s četiri preostale
dimenzije upitnika: kompetencija odraslih, otvorenost dječjeg vrtića,
filozofija inkluzije, kriteriji praćenja inkluzije. Ovaj podatak moguće je
protumačiti pretpostavkom da su predrasude prema inkluziji (koje su
veće u funkciji kronološke dobi) vjerojatno uvjetovane s jedne strane
većim znanjem o objektivnim organizacijsko-pravnim mogućnostima
i statusom struke (manje nerealnih očekivanja), a s druge strane od-
sustvom specifičnih znanja o inkluziji. ZAKLJUČAK Konstruiran je mjerni instrument sa sedam latentnih dimenzija
stavova prema inkluziji, koje pokazuju nisku ali granično zadovoljavaju-
ću pouzdanost. Pouzdanost pojedinih dimenzija stavova prema inklu-
ziji je za tri skale vrlo niska (tj. loša): organizacijska prilagodba, kriteriji
praćenja inkluzije, filozofija inkluzije. Za dimenzije kompetencija odra-
slih i otvorenost dječjeg vrtića pouzdanost je osrednje zadovoljavajuća,
tj. prihvatljiva, dok je za preostale dimenzije pouzdanost niska (upitna). Dobivene dimenzije pokrivaju nekolicinu područja edukacije o inkluziji,
pa podržavamo početnu hipotezu. Od ukupno 21 korelacije dimenzija
stavova prema inkluziji, 8 ih je većinom nisko, ali statistički značajno i
pozitivno povezanih. Dakle, podržavamo i hipotezu o postojanju pove-
zanosti između pojedinih dimenzija stavova prema inkluziji. Rezultati iz ovog istraživanja daju jasniji i precizniji uvid u pod-
ručja stavova odgojitelja- praktičara prema inkluziji, koja mogu biti
relevantna za tumačenje problematike provedbe inkluzije u uvjetima
redovitih dječjih vrtića. S praktičnog aspekta, nalaz ovog istraživanja
mogao bi značiti prvenstveno da možemo sustavno procjenjivati efek-
te edukacije o inkluziji: i u inicijalnom stanju (prije edukacije), i u final-
nom (nakon edukacije). Poželjne promjene stavova prema inkluziji (u
smislu boljeg razumijevanja inkluzije kao i načina njenog provođenja
u praksi, za dobrobit djeteta) kao rezultanta edukacije o inkluziji, od-
nosno odsustvo takvih poželjnih promjena stavova i prema inkluziji
može nam pomoći da korigiramo i prilagodimo edukacijski program. Međusobna povezanost dimenzija stavova prema inkluziji 330 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće Međusobna povezanost dimenzija stavova prema inkluziji Moguće je da sudionice mož-
da u većoj mjeri vrednuju područja koja obuhvaćaju spoznajni aspekt
(saznanja o inkluziji i uvjetima njena provođenja u dječjem vrtiću), ali
i stavove (organizacijsku prilagodbu, filozofiju inkluzije) te osobne i
stručne kompetencije, nego područja koja potencijalno otvaraju više 329 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. njihovih etičkih i afektivnih dilema, kao što su otvorenost dječjeg vr-
tića, predrasude prema inkluziji te kriteriji praćenja inkluzije. Pozitivni doprinos ovog istraživanja jest konstrukcija metrijski
zadovoljavajućeg instrumentarija (na temelju radne verzije upitnika
korištenog za praktične svrhe) za ispitivanje stavova odgojiteljica pre-
ma inkluziji, te za evaluaciju efekata edukacije o inkluziji. Međutim,
konstruirani upitnik tumači relativno mali postotak ukupne varijan-
ce, pa rezultati njegove primjene istovremeno mogu biti i koristan po-
datak o heterogenosti općih stavova odgojiteljica prema inkluziji, ali
i smjernica za daljnje usavršavanje upitnika. S druge strane, najveći
nedostatak istraživanja jest činjenica da je u odnosu na inicijalni broj
čestica upitnika, broj sudionica ipak bio relativno mali, što je moglo
uzrokovati dobivenu strukturu latentnih dimenzija (s manjim postot-
kom objašnjene varijance). Dimenzije koje smo dobili u ovom istraživanju su slične oni-
ma koje smo dobili u preliminarnom istraživanju (Kubelka i Sindik,
2009), jednostavnim grupiranjem čestica u odnosu na njihov smisao
(sadržajnu valjanost), dakle isključivo na temelju radne verzije upitni-
ka, sa znatno manjim brojem sudionica. Čak i bez obzira na utvrđene
manjkavosti istraživanja, ono ukazuje na mogućnost sustavnog pro-
cjenjivanja učinkovitosti edukacije o inkluziji na promjenu stavova,
ali i na mogućnost unaprjeđivanja kvalitete edukacijskog programa i
njegove prilagodbe ciljnoj skupini polaznika edukacije. Stoga bismo u budućnosti mogli provesti istraživanje na većem
uzorku sudionica, jer je moguće da bismo na većem uzorku sudionica
dobili drugačije metrijske karakteristike čestica, te dimenzije upitnika
u cijelosti. S druge strane, mogli bismo poboljšanja planirati i u teorij-
skom, i u metrijskom području: s teorijskog aspekta, moguće je redefi-
nirati područja edukacije, što bi vjerojatno rezultiralo i posve drugači-
jim dobivenim dimenzijama upitnika. S metrijskog stanovišta, mogli
bismo iskušati drugačije faktorske solucije, pa bismo, uz rigorozniji
kriterij saturacije faktora (primjerice, korelacija varijable s faktorom
veća ili jednaka 0,40), mogli doći do posve drugačijih dimenzija nego u
ovom istraživanju. Hipotetski, potencijalno bi se primjerice dimenzije
kompetencija odraslih te osobne i stručne kompetencije, mogle obje-
diniti kao nova dimenzija „kompetencije”, i slično. LITERATURA 1. Arsenijević-Puhalo, A., i Matošević, V. (2007). Rastimo zajedno
(stavovi zaposlenih u predškolskim ustanovama o inkluziji). Banja
Luka: Ombudsman Republike Srpske - Zaštita prava djece. 2. Bezuk-Jakovina, J. (2002). Roditeljstvo i suradnja. U L. Kiš-Glavaš
& R. Fulgosi-Masnjak (Ur.), Do prihvaćanja zajedno: Integracija djece
s posebnim potrebama. Priručnik za učitelje. (str. 180-197). Zagreb,
Hrvatska: Udruga za stručnu pomoć djeci s posebnim potrebama
- IDEM. 2. Bezuk-Jakovina, J. (2002). Roditeljstvo i suradnja. U L. Kiš-Glavaš
& R. Fulgosi-Masnjak (Ur.), Do prihvaćanja zajedno: Integracija djece
s posebnim potrebama. Priručnik za učitelje. (str. 180-197). Zagreb,
Hrvatska: Udruga za stručnu pomoć djeci s posebnim potrebama
- IDEM. 331 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. 3. Cerić, H. (2008). Mogućnost konstituiranja teorije inkluzivnog
obrazovanja. Metodički obzori, 3(1), 49-62. 3. Cerić, H. (2008). Mogućnost konstituiranja teorije inkluzivnog
obrazovanja. Metodički obzori, 3(1), 49-62. 4. Cheng, Y.L. (2001). Perceptions of Inclusion by Kindergarten
Teachers and Parents in Taiwan. Journal of Pingtung Teachers
College, 15, 259-292. 5. Chung, M. (2000). A study of early childhood teachers’ professional
knowledge and their perceived problems of inclusive classes. Taiwan:
National Changhua University of Education. Unpublished doctoral
dissertation. 6. Daniels, E.R., & Stafford, K. (2003). Kurikulum za inkluziju. Razvojno-primjereni program za rad s djecom s posebnim potrebama. Zagreb: Udruga roditelja Korak po korak za promicanje kvalitete
življenja djece i obitelji. 7. Donnelly, P., & Coakley, J. (2002). The role of recreation in promoting
social inclusion. Ottawa, Canada: The Laidlaw Foundation. Preuzeto
sa http://www.ccsd.ca/subsites/inclusion/bp/pd.htm 2003 Oct 17. 8. Državni pedagoški standard predškolskog odgoja i naobrazbe
(2008) Preuzeto sa http://cadial.hidra.hr/searchdoc.php?query=D
r%C5%BEavni+pedago%C5%A1ki+standard+pred%C5%A1kolsk
og+odgoja+i+naobrazbe&searchText=on&searchTitle=on&filterac
ttype=all&filtereuchapter=all&filterfields=all&resultlimit=on&r
esultlimitnum=10&annotate=on&displayOptions=on&lang=hr&
resultoffset=0&id_doc=gt26NBM7c89Uv%2b4slLdKUQ%3d%3d
2012 May 5. 9. Eiserman, W.D., Shisler, L., & Healey, S. (1995). A community
assessment of preschool providers’ attitudes toward inclusion. Journal of Early Intervention, 19(2), 149-167. 10. Freiler, C. (2001). What needs to change?, Towards a vision of social
inclusion for children, families and communities. Toronto, Ontario:
Laidlaw Foundation. Draft Concept Paper. 11. Gemmell-Crosby, S.J., & Reditti, H.J. (1994). Preschool teachers
perceptions of including children with disabilities. Education and
Training in Mental Retardation and Developmental Disabillities, 29(4),
279-290. 12. Kubelka, R., i Sindik, (2009). Utjecaj edukacije o inkluziji na pro-
mjenu stavova prema inkluziji. U: R. Babić & Z. Redžeš-Borak 332 Sindik, J.: Konstrukcija upitnika stavova odgojiteljica prema
inkluziji djece s teškoćama u razvoju u dječje vrtiće (Ur.), Zbornik radova 4. stručnog i znanstvenog skupa Dječji vrtić-
mjesto učenja djece i odraslih. LITERATURA Osijek: Centar za predškolski odgoj
i Učiteljski fakultet u Osijeku.179-188. (Ur.), Zbornik radova 4. stručnog i znanstvenog skupa Dječji vrtić-
mjesto učenja djece i odraslih. Osijek: Centar za predškolski odgoj
i Učiteljski fakultet u Osijeku.179-188. 13. Leš, A. (2005). Inkluzija djece s teškoćama u razvoju u redovan
sustav odgoja i obrazovanja. Preuzeto sa http://www.roda.hr/
tekstovi.php?TekstID=7&Tekst2ID=10&Show=1774 2012 May 5. 14. Levandovski, D., i Teodorović, B. (1991). Kako poticati dijete s men-
talnom retardacijom. Priručnik za roditelje. Zagreb: Fakultet za de-
fektologiju Sveučilišta u Zagrebu, Centar za rehabilitaciju Zagreb. 15. Mejovšek, M. (2003). Uvod u metode znanstvenog istraživanja. Za-
greb: Naklada Slap. 16. Mustać, V., i Vicić, M. (1996). Rad s učenicima s teškoćama u razvoju u
osnovnoj školi. Priručnik za prosvjetne djelatnike. Zagreb: Školska knjiga. 17. Nunnally, J.C. (1978). Psychometric theory. New York: McGraw-Hill. 18. Peck, C., Carlson, P., & Helmstetter, E. (1992). Parent and teacher
perceptions of outcomes for typically developing children enrolled
in integrated early childhood programs: A statewide survey. Journal of Early Intervention, 16(1), 53-63. 19. Programsko usmjerenje odgoja i obrazovanja predškolske djece (1991). Glasnik Ministarstva kulture i prosvjete Republike Hrvatske, 7/8. 20. Rose, D.F., & Smith, B.J. (1993). Preschool mainstreaming:
Attitude barriers and strategies for addressing them. Young Child,
48(4), 59-62. 21. Smith, D., & Luckasson, R. (1994). Introduction to special education:
Teaching in an age of challenge. Needham Heights, MA: Allyn and Bacon. 22. Stroeve, W. (1998). One of the Kids. Sdney: Disability Council of NSW 23. Stubbs, S. (1998). What is Inclusive Education?, Concept Sheet. Enabling Education Network (EENET). Preuzeto sa http://www. eenet.org.uk/theory_practice/whatisit.shtml 2003 Oct 16. 24. Stuber, G. (1994). Investigation of educators’ attitudes toward the
integration of young children with disabilities into regular kindergarten
classrooms. Lawrence: University of Kansas. 25. Zhao, N. (2009). The Minimum Sample Size in Factor Analysis. Preuzeto sa http://www.encorewiki.org/display/~nzhao/The+Min
imum+Sample+Size+in+Factor+Analysis 2012 May 5. 333 Specijalna edukacija i rehabilitacija (Beograd), Vol. 12, br. 3. 309-334, 2013. CONSTRUCTION OF QUESTIONNAIRE FOR
KINDERGARTEN TEACHERS ABOUT THE ATTITUDES
TOWARDS INCLUSION OF CHILDREN
WITH DISABILITIES Joško Sindik
Institute for Anthropological Research, Zagreb, Croatia Primljeno: 16.7.2013. Summary The main objective of this study was to construct an measuring
instrument for estimating the attitudes about inclusion. The sample
of predchool teachers from kindergartens in Osijek and Zagreb was
examined, using the questionnaire Atittudes of the experts about
the inclusion of children with development disabilities. We have
confirmed the existence of seven latent dimensions of attitudes about
the inclusion, defined as a simple linear combination of the items that
define each factor, showing low and marginally satisfactory reliability,
which ranges from very low (organizational adaptation, monitoring
the inclusion criteria) to moderately high. From all the correlations
between the dimensions of attitudes about inclusion, more than a third
of them are mainly low, but statistically significantly and positively
correlated. Based on the established metric characteristics, measuring
instrument can be considered sufficiently good initial starting point
for the construction of the final version of the instrument, intended
for the evaluation of training on inclusion of preschool children’s
teachers. Key words: metric characteristics, inclusion, preschool, attitudes,
factors of the attitudes Prihvaćeno: 8. 9.2013. Prihvaćeno: 8. 9.2013. Prihvaćeno: 8. 9.2013. Primljeno: 16.7.2013. 334
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Speed of Gravity: A Simple Experiment to Test the General Relativity Theory
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1. Introduction On the 14th of September 2020, LIGO celebrated the 5th Anniversary of the first Gravitational Wave detection. In the
celebration publicity, they projected what they have done within these five years including “the first measurement of the
speed of gravity confirming that gravity propagates at the speed of light” along with a dozen ‘firsts’ [1]. However, it seems
that by “gravity propagates” they mean “gravitational wave propagates”, but not the propagation of gravity action. Sergei Kopeikin and Edward Fomalont in 2002 claimed that they measured the speed of gravity and that was near the
speed of light which was not accepted by the physicists. In this article, we shall suggest a simple experiment that can measure the speed of gravity matching the seismic effect
and the fluctuation of solar activity as observed from the surface of the Earth. However, our suggestion is in the
preliminary stage. It requires a lot of assistance from the experts in this field to make the experiment practicable. Sankar Hajra sankarhajra@yahoo.com Qeios, CC-BY 4.0 · Article, December 7, 2023 Open Peer Review on Qeios Open Peer Review on Qeios Speed of Gravity: A Simple Experiment to Test the General
Relativity Theory Sankar Hajra1
1 Indian Physical Society Sankar Hajra1
1 Indian Physical Society Funding: No specific funding was received for this work. Potential competing interests: No potential competing interests to declare. Abstract In this article, the author has shown that LIGO-VIRGO’s Gravitational wave detection does not have any scientific
foundation. He has suggested a simple experiment (at a preliminary stage) that can detect the time- In this article, the author has shown that LIGO-VIRGO’s Gravitational wave detection does not have any scientific
foundation. He has suggested a simple experiment (at a preliminary stage) that can detect the time-
lagged/instantaneous seismic effect due to the fluctuation of solar activity to prove/disprove the General Theory of
Relativity. foundation. He has suggested a simple experiment (at a preliminary stage) that can detect the time
lagged/instantaneous seismic effect due to the fluctuation of solar activity to prove/disprove the General Theory of
Relativity. 2. Indirect Detection of Gravitational radiation (1994) In the last part of the twentieth century, American Scientist J. H. Taylor (Noble Lecture 1994, “Binary Pulsars and Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 1/9 Qeios, CC-BY 4.0 · Article, December 7, 2023 Relativistic Gravity”) showed that the calculated gravitational radiation loss of the binary pulsar PSR 1913+ 16 matches
well with the contraction of the orbit of that pulsar from which he claimed the existence of gravitational waves indirectly. However, Ehlers et al., some experts on the General Relativity Theory maintain that the problem of motion and
(gravitational) radiation (in the General Relativity Theory) was full of holes large enough to drive trucks through. C. M. Will, a strong supporter of the Special and General Relativity Theories thinks that the questions raised by Ehlers et
al. were still relevant: Is the quadruple formula for binary systems the actual prediction of the General Relativity
Theory [2]? Let us consider that a hypothetical binary pulsar is rotating in a stable circular orbit having a radius r=1.750×106 km with a
velocity u= 280 km/sec under the action of a gravitating force field. Suppose that the pulsar does not radiate. Now say a
small central electromagnetic force begins to act on the pulsar such that the orbital period (P) of the pulsar changes as
per the relation dP/dt=−2.4× 10-12. Let us calculate the ratio between the gravitating acceleration (fg) and the
electromagnetic acceleration (fem) of the pulsar. dP/dt= (2π/u) dr/dt=−2.4× 10-12; Therefore, dr/dt = fem =−1.07× 10-10 km /sec2:
fg= −u2/r= −4.48 × 10-2 km /sec2; Therefore, fg/ fem= 4.19× 108. dP/dt= (2π/u) dr/dt=−2.4× 10-12; Therefore, dr/dt = fem =−1.07× 10-10 km /sec2:
fg= −u2/r= −4.48 × 10-2 km /sec2; Therefore, fg/ fem= 4.19× 108. dP/dt= (2π/u) dr/dt=−2.4× 10-12; Therefore, dr/dt = fem =−1.07× 10-10 km /sec2:
fg= −u2/r= −4.48 × 10-2 km /sec2; Therefore, fg/ fem= 4.19× 108. dP/dt= (2π/u) dr/dt=−2.4× 10-12; Therefore, dr/dt = fem =−1.07× 10-10 km /sec2:
fg= −u2/r= −4.48 × 10-2 km /sec2; Therefore, fg/ fem= 4.19× 108. A minuscule EM acceleration approximately half a billionth part of the gravitational acceleration could change the orbital
period of the pulsar to the same extent as measured by Hulse and Taylor. A minuscule EM acceleration approximately half a billionth part of the gravitational acceleration could change the orbital
period of the pulsar to the same extent as measured by Hulse and Taylor. 2. Indirect Detection of Gravitational radiation (1994) However, if we could properly measure the electromagnetic radiation and take it into account, that acceleration should be
much less. Charge loss of the pulsar and consequent alteration of the electric and magnetic fields could decrease the
orbit to that minuscule amount. Pulsars are electromagnetic bodies. Orbits of electrodynamic bodies should not be stable from electrodynamic viewpoint. Therefore, pulsars are not ideal objects to study the relation of orbital decay with esoteric gravitation radiation. As per the current astrophysics, Gravitational radiation emission is decreasing the Earth’s orbit by a diameter of a proton
every day. LIGO claims that it could measure less than one-thousandth of the charge diameter of a proton. LIGO may
start a global call to attract a billion-dollar investment to prove Taylor’s contention for the orbital decay of the Earth and the
planets! But, at the present state of our knowledge, we could safely conclude that Taylor’s matching hardly proves Gravitational
wave. 3. Direct Detection (2016) In 1968, American physicist Joseph Weber claimed that he had detected gravitational waves using his detector made up
of enormous aluminium cylinders. This was sadly later disproved. Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 2/9 Qeios, CC-BY 4.0 · Article, December 7, 2023 In 2014 the scientists behind the BICEP 2 telescope made an extraordinary claim that they had detected gravitational
waves which are ripples in space-time. Initially hailed as the most ground-breaking discovery of the century, it was later
proved to be incorrect. In 2014 the scientists behind the BICEP 2 telescope made an extraordinary claim that they had detected gravitational
waves which are ripples in space-time. Initially hailed as the most ground-breaking discovery of the century, it was later
proved to be incorrect. Recently LIGO and VIRGO have claimed that they detected an instantaneous gravitational wave on the 14th of
September 2015 at 09:50:45 UTC in their two Michelson interferometers (separated by 3000 KM) simultaneously [3]. LIGO
Experimenters write that in their detection “signal sweeps upwards in frequency from 35 to 250 Hz with a peak
gravitational-wave strain of 1× 10-21.” It matches with the wave-form predicted by GR for inspiral and merger of a pair of
black holes (having masses 36+5
−4 M☉ and 29+4
−4 M☉ respectively) and the ringdown of the resulting single black hole
(having mass 62+4
−4 M☉ and gravitational radiation of 3−0.5
+0.5 M☉ c2) within a fraction of a second 1.3 billion years ago [3]. 3.1. Gravitational Wave Detection of LIGO-VIRGO and its Criticism from Technicalities Wolfgang W. Engelhardt, a LIGO dissident physicist who retired from the Max-Planck-Institut für Plasmaphysik, raised
some most pertinent questions on the technicalities of the experiment. Engelhardt writes on the Gravitational wave detection of LIGO, “In order to substantiate this extraordinary claim, it is
necessary to demonstrate experimentally LIGO’s ability to measure a displacement of 10-18 m that is one-thousandth of a
proton radius. The reader is assured that the calibration of the system can be achieved by moving the mirrors by such a
tiny distance with radiation pressure [4]. “Formula (10) gives the calculable connection between displacement and the radiation power of an auxiliary laser shining
on the mirror. 3. Direct Detection (2016) Unfortunately no data are given as to the laser power, wave form, number of oscillations in order to
compare with the documented effect that was exerted on the mirrors by the wave GW150914” as displayed in the
discovery paper [4]. “An inquiry with the Albert Einstein Institut revealed that such data do not exist” [4]. James Creswell et al. [5][6] of some reputable research institutes in Denmark, Hao Liu et al. [7], Brookes [8] Jackson et
al. [9], Sabine Hossenfelder [10] Alexander Unzicker[11] and many others [12] have not accepted LIGO’s claim. Creswell et al [6] opine Creswell et al. [6] opine, “The results presented here suggest this level of cleaning has not yet been obtained and that the detection of the
GW events needs to be re-evaluated with more careful consideration of noise properties.” Creswell et al. [6] also write, “We note, however, that a potential increase in the BBH masses would raise fundamental questions regarding their Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 3/9 Qeios, CC-BY 4.0 · Article, December 7, 2023 origin.” [BBH: Binary Black Hole]. According to Hao Liu et al. [7] “We have presented one such approach here and used it to analyze the data for GW150914. Cases in which the
signals are too weak to permit unbiased determination and which therefore require the use of templates for signal
detection should be regarded with extreme caution: It is likely that the conclusions of such analyses will be
determined by theoretical preconceptions and not by the data itself.” Brooks [8] comments: Brooks [8] comments: Brooks [8] comments: “The paper on the first detection used a data plot that was more ‘illustrative’ than precise.” He further adds, He further adds, “Jackson’s group says the decisions made during the LIGO analysis are opaque at best and probably wrong.” “Jackson’s group says the decisions made during the LIGO analysis are opaque at best and probably wrong.”
According to Jackson et al.[9], According to Jackson et al. [9], a truism that if gravitational waves are all you look for, gravitational waves are all you will ever find.” Lost in Mathematics author Sabine Hossenfelder [10] presently a Research Fellow at the Frankfurt Institute for Advanced
Studies, comments on the detection: “This gives me some headaches, folks. 3. Direct Detection (2016) If you do not know why your detector detects something that does not look
like what you expect, how can you trust it in the cases where it does see what you expect?” Alexander Unzicker [11] writes on the detection, “Nothing is more difficult than not to deceive yourself — Ludwig Wittgenstein.” “Nothing is more difficult than not to deceive yourself — Ludwig Wittgenstein.” Most of those objections are from the consideration of the technicalities of the experiment and these technicalities are
beyond the expertisation of most of the physicists. Criticisms cited above are on the technicalities of the experiment but
not on the physics of the experiment. 3.2. Physical Foundation of the LIGO-VIRGO Experiment on the Detection of Gravitational Wave
The scientists of LIGO-VIRGO believe that gravitational waves cause space itself to stretch in one direction and Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 4/9 Qeios, CC-BY 4.0 · Article, December 7, 2023 simultaneously compress in a perpendicular direction. This causes one arm of the Michelson interferometer to get longer
while the other gets shorter, and then vice versa, back and forth as long as a gravitational wave is passing towards the
interferometers. As the lengths of the arms change during the propagation of gravitational waves, so too do the distances traveled by each
laser beam. A beam in a shorter arm will return to the beam splitter before a beam in a longer arm. Then, the situation
reverses. So, when a gravitational wave passes through the Michelson Interferometer, its detector will register a fringe shift that
should be the measure of the strength of the passing gravitational wave. So, when a gravitational wave passes through the Michelson Interferometer, its detector will register a fringe shift that
should be the measure of the strength of the passing gravitational wave. The LIGO-VIRGO scientists assume that the speed of light in both the spaces that are steadily stretching and steadily
compressing is the same ‘c’ as that of light in free space. The LIGO-VIRGO scientists assume that the speed of light in both the spaces that are steadily stretching and steadily
compressing is the same ‘c’ as that of light in free space. According to Maxwell, the speed of light in free space is ‘c’ which is well-verified by experiments. SRT accepts this
proposition. 3. Direct Detection (2016) But this speed of light is fully dependent on the permeability and permittivity of the medium through which
light propagates. Now if free space is steadily compressed and steadily stretched, it is evident from physics that the permeability and
permittivity of that space must change and thereby the speed of light in that space should change if not otherwise proved
by experiment. Now if free space is steadily compressed and steadily stretched, it is evident from physics that the permeability and
permittivity of that space must change and thereby the speed of light in that space should change if not otherwise proved
by experiment. But it has not been proved to this day by experiments that the speed of light is the same ‘c’ in the stretched and
contracted spaces contrived by the relativists. But it has not been proved to this day by experiments that the speed of light is the same ‘c’ in the stretched and
contracted spaces contrived by the relativists. To justify the theoretical foundation of the LIGO-VIRGO experiments, LIGO-VIRGO experts should prove, through an
independent experiment, that the speed of light is indeed the same ‘c’ in those steadily stretching and compressing
spaces, just as it is in free space. They have not done this yet. Therefore, the GW detection process of the LIGO-VIRGO
does not have any scientific foundation [12]. This simple argument from physics nullifies the claim of LIGO-VIRGO instantly. No other arguments are seemingly
necessary to rubbish the claim further. 4. Speed of Gravity As to Newton, the action of gravitation is instantaneous, whereas as per Einstein, this action too travels with the speed of
light, and the latter consideration has been used by LIGO and VIRGO. However, whether the action of gravitation is
instantaneous or time-lagging has not been demonstrated in any reliable experiment just like many other propositions of
the relativists. Maxwell’s equations show that light travels with a speed of ‘c’ in the free space. This famous equation of Maxwell is called Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 5/9 Qeios, CC-BY 4.0 · Article, December 7, 2023 the homogeneous wave equation. This equation does not tell us anything about the speed of the electric and magnetic
fields in the free space. the homogeneous wave equation. This equation does not tell us anything about the speed of the electric and magnetic
fields in the free space. However, the Lorenz gauge condition which is generally used in the calculation of time-dependent electromagnetic fields
has a similar form to that of the homogeneous wave equation except the right-hand side of the equation not being 0. Therefore, Lorenz Gauge, sometimes called the inhomogeneous wave equation may imply that the speed of the electric
field/ magnetic field in free space is ‘c’. Any experiment in electrodynamics does not contradict this conclusion. Albert
Einstein accepts this conclusion. In the case of Newton’s law of gravity, the gravitational force acts instantly. No experiment to this day contradicts this
conclusion. No retarded effect of gravity action has been registered to this date. But many physicists believe that gravity should have speed and Albert Einstein thinks that gravity has the speed of ‘c’. One important point to remember here is that the electric field and the magnetic field are medium-dependent fields but
gravity acts independently of the type of medium. Therefore, we should not jump to any conclusion on the speed of gravity from the consideration of prevailing covariant
faith in physics. To know the truth a correct simple experiment free from mathematical jugglery should be done. To know the truth a correct simple experiment free from mathematical jugglery should be done. 5. A Suggested Experiment to know the Speed of Gravity We know that “Maximum quake frequency occurs at times of moderately high and fluctuating solar activity. Terrestrial
solar flare effects which are the actual coupling mechanisms that trigger quakes appear to be either abrupt accelerations
in the earth's angular velocity or surges of telluric currents in the earth's crust”[13]. Fluctuations in solar activity have many such effects on seismic activity, which could be studied accurately. The
fluctuations of solar activity could also be accurately and continuously photographed. Now, matching this fluctuation with
seismic activity, it is possible to know whether there is a time lag of 499 seconds between the physical effects and
electromagnetic effects propagated from the Sun to the Earth. However, I have received a practical suggestion from Jonathan Merrison of the Department of Physics and Astronomy,
Aarhus University in this regard that we are conveying to the readers. “An experimental determination of the speed of gravity (or change in gravity) I think would be extremely beneficial
to the science community. The issue is relevant and even controversial with regard to modern theories of gravity. If I understand correctly then the suggested experiment is NOT to use the LIGO/VIRGO GW instruments, but
instead to use terrestrial seismometers to sense solar flare arrival at Earth compared to light travel. It is not clear
whether the seismometers should directly measure the solar flare or sense some reaction by the Earth. Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 6/9 Qeios, CC-BY 4.0 · Article, December 7, 2023 I would suggest supporting this idea with some crude (rough order of magnitude) calculations which could indicate
whether some kind of sensors would be capable of detecting this. I have tried to do this myself below; by
comparing the effect with the solar tides which are detectable (maybe to around 1% accuracy), though I think not
with seismometers. I would suggest supporting this idea with some crude (rough order of magnitude) calculations which could indicate
whether some kind of sensors would be capable of detecting this. I have tried to do this myself below; by
comparing the effect with the solar tides which are detectable (maybe to around 1% accuracy), though I think not
with seismometers. Instead of a solar flare (which is low mass) I have taken a Coronal Mass Ejection event which can have a mass of
around 1012kg and velocity of around 106m/s (they have similar energy to a solar flare i.e. 5. A Suggested Experiment to know the Speed of Gravity Our suggestion is in the preliminary stage and we expect the contributions of experts in this field to make the experiment
successful. 5. A Suggested Experiment to know the Speed of Gravity around 1024 J). Taking a
time scale for the coronal mass ejection to be 100 seconds, it has travelled 108m. The tidal forces produced by the sun are measurable on the Earth (solar tides) and are related to the gradient in
the solar g (at earth) dgsun/dr = G. Msun/R3 where R is the distance to the sun and Msun is sun’s mass
(R=1.5x1011m, Msun=2x1030kg). For the solar tides the time scale is around 6 hrs (between tides). Comparing the effect of a Coronal Mass Ejection to the solar tides we see that the distance change is around 10-3
(r/R or even (R-r)3 compared to R3) the time is around x200 faster, but the dominant factor is that the mass is 10-
18 smaller. This is very small. If it was 10-3 or even 10-6 then I think that it might be measurable, but I would expect
this not to be measurable unless there is something that I am not understanding. However, if you are suggesting that the LIGO/VIRGO GW instruments try to look for a Coronal Mass Ejection then
I think that this idea might have a valid argument. If I again try to perform a rough order of magnitude calculation to
compare the claimed GW signal (from the assumed black holes) then I find this, (note that I am just guessing
values here I am not using proper values obtained by them); Assume distance to observed BBH = 5GPc =1026 m, masses involved =103 Msun, velocities involved v <c < 108
m/s. Assume distance to observed BBH = 5GPc =1026 m, masses involved =103 Msun, velocities involved v <c < 108
m/s. Assuming the signal deceases as Mv/r2 (r distance to the GW generator) then the; Signal from Coronal Mass Ejection/Signal from BBH = (1012/1033). (106/108)/(1011/1026)2 = 10-21.10-2.1030 = 107 So a Coronal Mass Ejection should give a much bigger signal (107 times bigger), though I think actually the GW
signal is a dipole effect and should decrease as 1/r3 but this would make the factor even larger. Obviously, some
of my assumptions may be flawed, but maybe you could perform a similar calculation to support your idea”. Our suggestion is in the preliminary stage and we expect the contributions of experts in this field to make the experiment
successful. 6. Conclusion Electromagnetic fields, electric charges and light possess momenta and energies that we can experience with our sense
organs. Therefore, these are real physical entities (objects). Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 7/9 Qeios, CC-BY 4.0 · Article, December 7, 2023 All objects are subject to gravitation and they are carried with the Earth at the near vicinity of its surface, and they
experience Coriolis force when they are part of the Earth System and move with respect to that system. All objects are subject to gravitation and they are carried with the Earth at the near vicinity of its surface, and they
experience Coriolis force when they are part of the Earth System and move with respect to that system. All electromagnetic entities do act similarly and this simple consideration is equivalent to the Special and General
Relativity Theory of Albert Einstein [14][15]. Many physicists all over the world are challenging the ultra-mundane validity of Einstein’s Physics. Tounsi [16] does not
believe in the equivalence principle operating for the nano-bodies. Spavieri [17] does not accept that the one-way speed of
light is ‘c’, Stephen Crothers exposes the flaws of the Special and General relativity. Cynthia K. Whitney thinks that
“Newton was surely wrong to insist that gravitational action is instantaneous. But Einstein was probably too casual in
suggesting that the speed of gravitational action is like the speed of light”. Physical Interpretation Relativity Theory London organised by the Late Dr M.C. Duffy published numerous papers
challenging the foundation of the relativity theory. Many dedicated rational physicists associated with David de Hilster’s
John Chappell Natural Philosophy Society along with Roger Anderton, Dennis P. Allen Jr., and many others have been
conducting weekly discussions on the fallacies of the Relativity Theory regularly. Penniless rational physics is fighting hard against the billion-dollar phantom physics. We are confident of the victory of rationality in physics shortly. Phantom physics must not survive. Acknowledgments The author is grateful to A. Kaci, C. Poitou, M. Cartmell, M. Figliolia, C. Frajuca, J. Merrison, G.Spavieri, E. Porcelli and for their kind suggestions to improve the article. References 1. ^Burtnyk, Kimberly (2020): LIGO Celebrates 5th Anniversary of First Gravitational Wave Detection. News Release,
September 14, 2020. https://www.ligo.caltech.edu/news/ligo20200914 1. ^Burtnyk, Kimberly (2020): LIGO Celebrates 5th Anniversary of First Gravitational Wave Detection. News Release,
September 14, 2020. https://www.ligo.caltech.edu/news/ligo20200914 2. ^Will, Clifford, M. The confrontation between general relativity and experiments, Living Rev. Relativity 17:4 58 (2014)
3. a, bAbbott, B. P., Abbott, R., Abbott, T. D., et al.. (2016). Observation of Gravitational Waves from a Binary Black Hole
Merger. Physical Review Letters 116(6) 061102. https://doi.org/10.1103/PhysRevLett.116.061102 4. a, b, cEngelhardt, Wolfgang (2026). LIGO: Wolfgang Engelhardt’s Letter to the Noble Committee. The Dreamheron
Chronicle. https://dreamheron.wordpress.com/2016/07/22/ligo-wolfgang-engelhardts-letter-to-nobel-committee/
5. ^Creswell, J., Von Hausegger, S., Liu, H., Naselsky, P., & Jackson, A. D. (2017). On the time lags of the LIGO signals. Journal of Cosmology and Astroparticle Physics 08 013. 4. , , Engelhardt, Wolfgang (2026). LIGO: Wolfgang Engelhardt s Letter to the Noble Committee. The Dreamheron
Chronicle. https://dreamheron.wordpress.com/2016/07/22/ligo-wolfgang-engelhardts-letter-to-nobel-committee/
5. ^Creswell, J., Von Hausegger, S., Liu, H., Naselsky, P., & Jackson, A. D. (2017). On the time lags of the LIGO signals. Journal of Cosmology and Astroparticle Physics 08 013. 5. ^Creswell, J., Von Hausegger, S., Liu, H., Naselsky, P., & Jackson, A. D. (2017). On the time lags of the LIGO signals. Journal of Cosmology and Astroparticle Physics 08 013. 6. a, b, cCreswell, J., Liu, H., Jackson, A. D., Von Hausegger, S., & Naselsky, P. (2018). Degeneracy of gravitational
waveforms in the context of GW150914. Journal of Cosmology and Astroparticle Physics 03 007. 6. a, b, cCreswell, J., Liu, H., Jackson, A. D., Von Hausegger, S., & Naselsky, P. (2018). Degeneracy of gravitational
waveforms in the context of GW150914. Journal of Cosmology and Astroparticle Physics 03 007. 6. a, b, cCreswell, J., Liu, H., Jackson, A. D., Von Hausegger, S., & Naselsky, P. (2018). Degeneracy of gravitational
waveforms in the context of GW150914. Journal of Cosmology and Astroparticle Physics 03 007. Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 8/9 Qeios, CC-BY 4.0 · Article, December 7, 2023 7. a, bLiu, H., Creswell, J., Von Hausegger, S., Jackson, A. D., & Naselsky, P. (2018). A blind search for a common signal
in gravitational wave detectors. Journal of Cosmology and Astroparticle Physics 02 013. 8. a, bBrooks, M. (2018). Exclusive, Grave doubts over LIGO's discovery of gravitational waves. New Scientist. https://www.newscientist.com/article/mg24032022-600-exclusive-grave-doubts-over-ligos-discovery-of-gravitational-
waves/ 9. a, bJackson, A. D., Liu, H., & Naselsky, P. (2019). Qeios ID: E1RS6Y.3 · https://doi.org/10.32388/E1RS6Y.3 References Noise residuals for GW150914 using maximum likelihood and
numerical relativity templates. ArXiv: 1903.02401v3 [astro-ph.IM], May 13, 2019. 10. a, bHossenfelder, S. (2019). What’s up with LIGO? Blog Back Reaction. https://backreaction.blogspot.com/2019/09/whats-up-with-ligo.html 11. a, bUnzicker, A. (2020). Gravitational Waves: The Silent Disaster. Blog. https://medium.com/swlh/gravitational-waves-
the-silent-disaster-ab18857c68f8 12. a, bHajra, S. (2023). Light speeds in stretching and contracting spaces. Qeios. https://doi.org/10.32388/JW6C3N 13. ^Simpson, J. F. (1967): Solar activity as a triggering mechanism for earthquakes. Earth and Planetary Science Letters
3, 417-425. Earth and Planetary Science Letters, 3, 417-425. 14. ^Hajra, S. (2022). An Attempt of Linking Maxwell with Newton to Study Electrodynamic Phenomena. Bulgarian Journal
of Physics 49 145–162. 15. ^Hajra, S. (2022). An Attempt of Linking Maxwell with Newton to Study Gravitational Phenomena. Bulgarian Journal of
Physics 49 314-328. 16. ^Tounsi, Abdelouahed (2022): On the new fifth fundamental force at the nanoscale. DOI:
10.13140/RG.2.2.35302.29769 17. ^Spavieri, G. and Haug, E. Paradigm shift in Special Relativity: From the Michelson-Morley experiment, Lorentz and
light speed invariance, to the reciprocal linear Sagnac effect and conservation of simultaneity. Queios, 2023. https://doi.org/10.32388/95U7HM 9/9
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Following Microstructures during Deformation: In situ X-ray/Neutron Diffraction and HRDIC
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40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 40th Risø International Symposium on Material Science E Polatidis1,2, K Sofinowski1,3, W-N Hsu1,3, H Van Swygenhoven1,3 E Polatidis1,2, K Sofinowski1,3, W-N Hsu1,3, H Van Swygenhoven1,3
1 Photons for Engineering and Manufacturing Group, Swiss Light Source, Paul
Scherrer Institute, Villigen-PSI 5232, Switzerland
2 Currently: Laboratory for Neutron Scattering and Imaging (LNS), Paul Scherrer
Institute, Villigen-PSI 5232, Switzerland
3 Neutrons and X-rays for Mechanics of Materials, IMX, Ecole Polytechnique
Federale de Lausanne, CH-1012 Lausanne, Switzerland 3 Neutrons and X-rays for Mechanics of Materials, IMX, Ecole Polytechnique
Federale de Lausanne, CH-1012 Lausanne, Switzerland Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd
1 helena.vanswygenhoven@psi.ch Abstract. The mechanical behavior of three engineering materials is studied employing in situ
deformation methods. The study covers metastable austenitic steels with different stacking fault
energies during multiaxial loading, a Ti-6Al-4V alloy processed by electron beam melting during
uniaxial deformation and a commercial nanocrystalline NiTi alloy during multiaxial
deformation. The experimental results obtained by in situ X-ray or neutron diffraction elucidate
the load transfer and phase transformation mechanisms, information that is averaged over a
relatively large volume containing a statistically representative number of grains. Complementary in situ high resolution digital image correlation allows details to be revealed
regarding the localized strain accommodation and slip activity with a sub-grain spatial
resolution. It is demonstrated that the synergy of the different length-scale investigations
provides a better understanding of the complex relationship between microstructure and
deformation behavior in these materials. Following Microstructures during Deformation: In situ X-
ray/Neutron Diffraction and HRDIC E Polatidis1,2, K Sofinowski1,3, W-N Hsu1,3, H Van Swygenhoven1,3 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 1. Introduction In situ neutron and synchrotron x-ray diffraction are well established techniques for studying internal
stress and microstructure evolution [1–3]. The evolution of diffraction peak positions, width and
intensity can provide insight on the average intergranular and intragranular strains, and texture evolution
within different grain families [4–8], where these grain families are classified according to their
crystallographic orientation with respect to the diffraction vector. The average lattice strain of a grain
family represents the fraction of applied load, i.e. type-I or macroscopic stresses, shared by that grain
family. Elastic anisotropy, plastic slip, grain neighborhood interactions and the direction of loading
significantly influence this evolution. In situ diffraction is also particularly useful for studying multi-
phase materials during deformation, as it reveals the load transfer between the phases and provides
information on the role of each phase for the strain hardening behavior of the composite material. The
technique has the advantage of providing average information over relative large volumes that are
representative of the studied microstructure. It misses, however, direct spatial microstructure
correlations, i.e. observations in individual grains, grain interactions or strain concentrations in
particular places. 1 40th Risø International Symposium on Material Science OP Conf. Series: Materials Science and Engineering 580 (2019) 012010
doi:10.1088/1757-899X/580/1/0 To have a better link with the microstructure, often electron backscatter diffraction (EBSD)
measurements are carried out before and after an in situ deformation and diffraction experiment. Such
studies provide additional information on the change of the microstructure in terms of deformation
texture, grain refinement or the formation of sub-grain boundaries. However, these observations only
reveal the microstructure in stationary states before and after the deformation, and do not provide
information on where and how strain is accommodated. Digital image correlation (DIC) is an optical method that employs tracking and image-registration
techniques and is used to follow changes in images. The method is employed in the engineering
community for measuring full-field displacement and strains. When applied at higher magnifications,
DIC of images obtained during mechanical testing makes it possible to study the localization of plastic
deformation on the surface of specimens. Coupling of DIC with scanning electron microscopy (SEM)
enables significantly more precise data to be obtained down to the submicron scale [9]. 1. Introduction The spatial
resolution that can be obtained depends to a great extent on the surface quality of the sample and on the
pattern that is used, for instance by using nano-sized microstructural independent patterns such as gold
speckle patterns [9] or particles from a colloidal silica suspension [10]. p
p
p
p
In situ high resolution digital image correlation (HRDIC) can offer a direct link between the
microstructure and the strain produced by dislocation slip, twinning and martensitic phase
transformations when the evolution during deformation is monitored in the same material portion. Such
localized information complements the average information obtained from diffraction techniques. Apart
from complimenting macroscopic experimental results, the strain localization obtained by HRDIC can
be coupled with crystal plasticity simulations [11]. Here we show that by using a miniaturized multiaxial deformation rig, implementable in an SEM, in
situ HRDIC can be carried out to capture the details of the strain hardening mechanisms that are missing
to explain the averaged load sharing and the phase transformation behavior obtained by in situ
synchrotron X-ray/neutron diffraction techniques. The cases illustrated are the transformation induced
plasticity (TRIP) effect in a 304 austenitic steel, the extraordinary hardening behavior in a Ti-6Al-4V
alloy processed by electron beam melting (EBM) and the multiaxial mechanical loading behavior of a
commercial superelastic NiTi alloy. 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 combination with a 20 μm aperture is used for the image acquisition. The images are acquired with an
in-lens detector at a working distance of approximately 7.5 mm in order to minimize the topographic
contrast by gathering low energy electrons, providing a good signal/noise ratio. SEM images are taken
at various magnifications depending on the size of the deformation features to be tracked with a
resolution of 3072 × 2304 pixels at the center of the cruciform/dogbone sample after reaching certain
strains determined based on the aim of the study. The HRDIC data is analyzed with Ncorr [18], using
typically a subset radius of 15 pixels, 0 subset spacing and a strain radius of 3 pixels, determining the
circle in which the strain is calculated. Pseudo-strains due to thermal drifts and lens distortion are
negligible compared to the strain magnitude observed in e.g. slip bands and therefore, no correction is
applied. combination with a 20 μm aperture is used for the image acquisition. The images are acquired with an
in-lens detector at a working distance of approximately 7.5 mm in order to minimize the topographic
contrast by gathering low energy electrons, providing a good signal/noise ratio. SEM images are taken
at various magnifications depending on the size of the deformation features to be tracked with a
resolution of 3072 × 2304 pixels at the center of the cruciform/dogbone sample after reaching certain
strains determined based on the aim of the study. The HRDIC data is analyzed with Ncorr [18], using
typically a subset radius of 15 pixels, 0 subset spacing and a strain radius of 3 pixels, determining the
circle in which the strain is calculated. Pseudo-strains due to thermal drifts and lens distortion are
negligible compared to the strain magnitude observed in e.g. slip bands and therefore, no correction is
applied. 2.3. Patterns for DIC f
The remodeling of a sputtered gold film can produce nanoscale aggregates of gold particles that can
serve as patterns for HRDIC analysis, resulting in strain mapping with sub-micron resolution [9,19]. As
a flat and well-polished surface is required in order to achieve good speckle patterns, the NiTi and Ti-
6V-4Al samples are ground and polished until a mirror-like surface is achieved. For the 304 steel
cruciforms, the novel fabrication process incorporating electrochemical micromachining results in a
high-quality surface, ready for direct application of the gold speckle pattern [20]. By adapting the
method proposed in [9], fine gold speckle patterns are applied to commercial superelastic NiTi and 304
steel to study the strain accommodation in commercial NiTi and the slip activity in 304 steel under
multiaxial deformation. An image of these patterns is shown in figure 1a and figure 1b. Figure 1. Fine gold speckle patterns allow HRDIC with a sub-micron resolution for (a) commercial
superelastic NiTi and (b) austenitic stainless steel 304. Figure 1. Fine gold speckle patterns allow HRDIC with a sub-micron resolution for (a) commercial
superelastic NiTi and (b) austenitic stainless steel 304. 3. Metastable austenitic steels under multiaxial loading 2.1. In situ multiaxial deformation f
For the in situ multiaxial loading and diffraction experiments, cruciform-shaped samples have been
designed with their geometry optimized with the aid of finite element analysis [12]. In situ synchrotron
X-ray diffraction experiments have been carried out at the material science beamline (MS) of the Swiss
synchrotron (SLS). The miniaturized multiaxial deformation rig [13] or a uniaxial tensile rig [14] are
mounted on the goniometer of the powder diffraction station at the MS beamline. The in situ
measurements are conducted in transmission mode using a Mythen II microstrip detector with counting
time of 30-60 seconds, depending on the material. More details about the experimental setups at the MS
beamline and the cruciform sample geometry are given in [14,15]. The multiaxial load rig available at POLDI is used for in situ neutron diffraction studies [16]. The
biaxial deformation system is equipped with a 2-camera digital image correlation (DIC) system (GOM,
Aramis 5M) for measuring the in-plane strain at the center of the cruciform-shaped sample. Uniaxial
loading and equibiaxial loading are performed with a loading rate of 80 N/s. The neutron diffraction
measurements are carried out in predefined force intervals after interrupting the loading and holding the
displacement. Typically the counting times range from approximately 30 minutes to 120 minutes
depending on the required accuracy of the diffraction information. The neutron diffraction data is
reduced and fitted using the open source software Mantid [17]. For the in situ HRDIC measurements, the miniaturized multiaxial deformation rig is installed inside the
chamber of a FEG SEM Zeiss ULTRA 55 [13]. The electron beam with acceleration voltage of 3 kV in 2 40th Risø International Symposium on Material Science 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 ε-martensite plates are preferred nucleation sites for α΄-martensite. The combined effect of the
crystallographic orientation and the load path on the strain-induced martensitic transformation is
rationalized as described in the following. The leading and trailing partial dislocations have different
Burgers vectors. In low SFE steels for grains with crystallographic orientation such that the leading
partial dislocation experiences a higher resolved shear stress than the trailing partial dislocation, the
partials separate and form an extensive stacking fault (SF), which allows the local formation of ε-
martensite, [23,24]. ε-martensite plates are preferred nucleation sites for α΄-martensite. The combined effect of the
crystallographic orientation and the load path on the strain-induced martensitic transformation is
rationalized as described in the following. The leading and trailing partial dislocations have different
Burgers vectors. In low SFE steels for grains with crystallographic orientation such that the leading
partial dislocation experiences a higher resolved shear stress than the trailing partial dislocation, the
partials separate and form an extensive stacking fault (SF), which allows the local formation of ε-
martensite, [23,24]. The opposite behavior is seen for 304 steel, i.e. more martensite is formed under biaxial than under
uniaxial deformation [22,25], as apparent by the diffraction peaks corresponding to martensite which
appear in figure 2-b, after approximately 15% von Mises strain. Under uniaxial deformation, no
diffraction peaks corresponding to martensite are seen until the end of the deformation, at approximately
27% strain, as shown in figure 2-a. Instead, a typical deformation behavior by slip is seen, where a strong
<111>-texture forms, parallel to the loading direction, accompanied by significant peak broadening, due
to dislocation activity, as seen in figure 2-a. Post mortem EBSD investigations show that the majority
of the grains under uniaxial deformation favor the formation of SFs (similarly to 201 steel) which in the
case of the higher SFE material, leads to the formation of deformation twinning. A significant number
of twins was observed by EBSD in favorably orientated grains under uniaxial deformation [22]. Figure 2. The evolution of the neutron diffracted intensities with increasing strain for a) uniaxial b)
equibiaxial tensile deformation of 304 steel. The neutron scattering vector is parallel to the loading
direction under uniaxial deformation and parallel with one of the loading axes under equibiaxial
deformation. 3. Metastable austenitic steels under multiaxial loading g
The deformation mechanisms under uniaxial and biaxial load paths were studied in two types of
metastable austenitic stainless steels, namely a low staking fault energy (SFE) 201 steel and a medium
SFE 304 steel. Both materials are fully austenitic, i.e., have a face-centred cubic crystal structure in the
unloaded state and exhibit martensitic phase transformation upon deformation. The evolution of the
phases was followed during in situ uniaxial and equibiaxial deformation by neutron diffraction. More
details on the experiments are given in [21,22]. In situ neutron diffraction performed on the low SFE 201 steel showed that for the same von Mises
strain, more martensite is formed under uniaxial tensile loading than under biaxial tensile deformation. Following the evolution of the neutron diffraction patterns with increasing strain, it is clear that ε-
martensite, which has a hexagonal closed packed crystal structure, forms as an intermediate phase prior
to the body centered α΄-martensite [21]. Post-mortem EBSD investigations show that the majority of
the grains under uniaxial deformation favour the formation of ε-martensite, and that the intercepts of the 3 40th Risø International Symposium on Material Science IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
doi:10.1088/1757-899X/580/1/012010 Thanks to the gold speckle pattern, the evolution of the slip activity within each grain can be followed
at different strain levels, as shown in figure 3-b and figure 3-c). Careful examination of 21 grains in each
loading state shows that equibiaxial deformation promotes the activation of slip on multiple slip planes
in the {111} lattice plane family within each grain, as shown in figure 3-e. Fewer grains with multiple
activated slip planes are observed under uniaxial loading (figure 3-d). It is thus concluded that the
enhanced twinning under uniaxial tensile deformation retards the activation of slip in secondary slip
planes, which in turn results in less martensite nucleation (as a consequence of the reduction in the
number of shear band intercepts, which are favorable nucleation sites for martensite formation). In
contrast, under equibiaxial tensile deformation, less twinning results in more martensite formation via
the activation of slip on more than one slip plane in each grain. Figure 3. (a) A miniaturized cruciform-shaped 304 steel sample with reduced thickness in the centre,
ready to be used for EBSD and HRDIC; (b-c) HRDIC showing slip traces at the grain level during
deformation under uniaxial and equibiaxial tensile deformation. The isolated grains (indicated with
red squares), show examples where a second slip system is activated. The statistical analysis (percent
of grains) shows that less grains exhibit multiple slip under (d) uniaxial than (e) equibiaxial
deformation [20]. (b)
Equibiaxial
Global 2% Global 6% Global 10% Global 14%
Global 2%
Global 6%
Global 10%
Global 14%
Uniaxial
Global 6%
Global 10%
Global 14%
Global 2%
15 μm
3 mm
100 μm
100 μm
(a)
(c)
15 μm
38.1%
61.9%
Single slip
Multi slip
57.1%
42.9%
10 μm
Global 14%
Global 14%
(d)
(e) 3 mm
100 μm
100 μm
(a) Global 2% Global 6% Global 10% Global 14% 10 μm 38.1%
61.9%
(d) (d) Single slip
Multi slip
57.1%
42.9%
(e) (e) Figure 3. (a) A miniaturized cruciform-shaped 304 steel sample with reduced thickness in the centre,
ready to be used for EBSD and HRDIC; (b-c) HRDIC showing slip traces at the grain level during
deformation under uniaxial and equibiaxial tensile deformation. The isolated grains (indicated with
red squares), show examples where a second slip system is activated. The statistical analysis (percent
of grains) shows that less grains exhibit multiple slip under (d) uniaxial than (e) equibiaxial
deformation [20]. 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 0
5
10
15
20
25
2.9
3.0
3.1
4.8
5.0
5.2
5.4
von Mises strain (%)
q (Å-1)
{211}BCC
{110}BCC
{220}FCC
{111}FCC
0
5
10
15
20
25
2.9
3.0
3.1
4.8
5.0
5.2
5.4
{220}fcc
{111}fcc
von Mises strain (%)
q (Å-1)
0
5
10
15
20
25
2.9
3.0
3.1
4.8
5.0
5.2
5.4
{220}fcc
{111}fcc
von Mises strain (%)
q (Å-1)
50.00
100.0
150.0
200.0
250.0
300.0
350.0
Intensity
(a.u.)
(a)
(b) 0
5
10
15
20
25
2.9
3.0
3.1
4.8
5.0
5.2
5.4
{220}fcc
{111}fcc
von Mises strain (%)
q (Å-1)
(a) 0
5
10
15
20
25
2.9
3.0
3.1
4.8
5.0
5.2
5.4
von Mises strain (%)
q (Å-1)
{211}BCC
{110}BCC
{220}FCC
{111}FCC
0
5
10
15
20
25
2.9
3.0
3.1
4.8
5.0
5.2
5.4
{220}fcc
{111}fcc
von Mises strain (%)
q (Å-1)
50.00
100.0
150.0
200.0
250.0
300.0
350.0
Intensity
(a.u.)
(b) (b) Figure 2. The evolution of the neutron diffracted intensities with increasing strain for a) uniaxial b)
equibiaxial tensile deformation of 304 steel. The neutron scattering vector is parallel to the loading
direction under uniaxial deformation and parallel with one of the loading axes under equibiaxial
deformation. The interpretation of the different amount of strain-induced α΄-martensite under uniaxial and
equibiaxial tensile deformation in 201 steel is evident by the suppression or facilitation of the formation
of the intermediate phase, i.e. ε-martensite. However, in the medium SFE 304 steel, the formation of
deformation twinning and its interaction with the martensite formation is not sufficiently understood to
explain the opposite behavior, i.e., why less transformation under uniaxial deformation than under
biaxial deformation is observed. Here HRDIC is a particularly useful tool for understanding the interplay
between deformation by slip, twinning and martensite formation in these materials, by following the slip
activity at the grain level. Figure 3-a shows an image of the miniaturized cruciform-shaped 304 steel sample and its surface
quality. For this study, a novel design for miniaturized cruciform samples was developed where the
center of the cruciform is thinned by electrochemical machining (figure 3-a), as described in [20]. The
sample surface can be readily used for electron backscatter diffraction (EBSD) and after applying a gold
speckle pattern, high resolution digital image correlation (HRDIC) can be carried out. 4 40th Risø International Symposium on Material Science IOP Publishing 4. Ti-6Al-4V The source of high-work hardening behavior was investigated for a Ti-6Al-4V alloy processed by EBM
and a novel post heat treatment. The samples were hot isostatic pressed (HIPed), annealed for two hours
at sub-β transus temperatures and then water quenched. The resulting microstructure consisted of large
α lamellae surrounded by regions of acicular α'-martensite, with the volume fraction of the martensite
regions depending on the annealing temperature. A full description of the preparation techniques is
provided in [26,27]. The post-treated material exhibits a higher ultimate tensile strength and, in some
cases, a higher ductility than the standard dual- phase α+β material obtained in the as-HIPed (no post
treatment) condition. This was found to be due to a high work hardening in the post-treated alloy, 2-3
times higher than in the as-HIPed samples [27]. The load sharing between the phases was investigated
with in situ XRD at the MS beamline (Swiss Light Source) and the results were corroborated with 5 40th Risø International Symposium on Material Science IOP Publishing 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 y p
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
g
doi:10.1088/1757-899X/580/1/012010 transmission electron microscopy (TEM), transmission Kikuchi diffraction (TKD), and in situ HRDIC
[28]. Figure 4. X-ray diffraction of the {10.2} (, {10.2} (′, and a Phase-3 peaks for Ti-6Al-4V at (a) 0
MPa (initial, unloaded state), (b) 800 MPa, and (c) 991 MPa,(d) the elastic lattice strain as function
of the applied stress( diffraction vector *+ perpendicular to the loading direction) transmission electron microscopy (TEM), transmission Kikuchi diffraction (TKD), and in situ HRDIC
[28]. sion electron microscopy (TEM), transmission Kikuchi diffraction (TKD), and in situ HRDIC transmission electron microscopy (TEM), transmission Kikuchi diffraction (TKD), and in situ HRDIC
[28]. Figure 4. X-ray diffraction of the {10.2} (, {10.2} (′, and a Phase-3 peaks for Ti-6Al-4V at (a) 0
MPa (initial, unloaded state), (b) 800 MPa, and (c) 991 MPa,(d) the elastic lattice strain as function
of the applied stress( diffraction vector *+ perpendicular to the loading direction) In situ uniaxial tensile loading experiments with XRD revealed the existence of a third phase in the
martensitic regions, mechanically distinct from the α and α' phases (figure 4). 4. Ti-6Al-4V The XRD peaks of this
phase are visible as shoulders at several HCP reflections before loading, and from additional peaks that
initially are obscured by nearby α reflections but which appear during loading as the phases take load
differently. This is shown in figure 4-a to c. The diffracted peaks could be fit as either a secondary
hexagonal closed packed (hcp) phase (secondary α) or an orthorhombic phase (α''-martensite) and could
not be identified with full confidence. Therefore, the phase will be henceforth referred to as "Phase-3."
The martensitic regions therefore consist of small laths of both α' and Phase-3. They are a few 100 nm
long, approximately one order of magnitude smaller than the α lamellae. The deformation is the result
of the load-sharing between the three phases, shown in figure 4-d for the peaks near the {10.2},-. reflection of a sample heat treated at 900°C. Note that the diffraction vector Q is perpendicular to the
loading direction, i.e. the calculated elastic lattice strains measure the Poisson contraction of the grain
families. Thus, in figure 4-d, a more negative value of the lattice strain implies that the sample takes
more load along the loading direction. In situ uniaxial tensile loading experiments with XRD revealed the existence of a third phase in the
martensitic regions, mechanically distinct from the α and α' phases (figure 4). The XRD peaks of this
phase are visible as shoulders at several HCP reflections before loading, and from additional peaks that
initially are obscured by nearby α reflections but which appear during loading as the phases take load
differently. This is shown in figure 4-a to c. The diffracted peaks could be fit as either a secondary
hexagonal closed packed (hcp) phase (secondary α) or an orthorhombic phase (α''-martensite) and could
not be identified with full confidence. Therefore, the phase will be henceforth referred to as "Phase-3."
The martensitic regions therefore consist of small laths of both α' and Phase-3. They are a few 100 nm
long, approximately one order of magnitude smaller than the α lamellae. The deformation is the result
of the load-sharing between the three phases, shown in figure 4-d for the peaks near the {10.2},-. reflection of a sample heat treated at 900°C. Note that the diffraction vector Q is perpendicular to the
loading direction, i.e. the calculated elastic lattice strains measure the Poisson contraction of the grain
families. IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
doi:10.1088/1757-899X/580/1/012010 IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
doi:10.1088/1757-899X/580/ Initially, the sample is in the elastic regime and the elastic lattice strains for all phases evolve linearly. At around 300 MPa, the sample enters a microplastic regime where the elastic lattice strains for α' and
Phase-3 deviate from linearity and begin to take less load. The α' peak broadens, suggesting it is
deforming plastically, while the peak width of the observable Phase-3 peaks does not change. Load is
transferred to the α phase. The onset of microplastic yield corresponds to the onset of plastic deformation
in several soft α grain families, which rapidly shed load. The α' grains continue to deform plastically
during this regime, and the load is transferred to the harder α grain families and Phase-3, which behaves
as the hardest phase. The presence of an additional hard phase allows the α and α' phases to deform
plastically beyond what they could in a dual-phase material, resulting in the enhanced work hardening
and ductility. y
Figure 5. In situ HRDIC of (a,b,c) OPS and (d,e,f) gold speckle pattern on Ti-6Al-4V (heat treated
at 900°C). Initial microstructures are backscatter electron images. Dark grains are α lamellae, light
gray regions are martensitic regions. Grain boundaries are drawn manually as a guide for the eye. Figure 5. In situ HRDIC of (a,b,c) OPS and (d,e,f) gold speckle pattern on Ti-6Al-4V (heat treated
at 900°C). Initial microstructures are backscatter electron images. Dark grains are α lamellae, light
gray regions are martensitic regions. Grain boundaries are drawn manually as a guide for the eye. To better understand the composite behavior of the martensitic regions, in situ HRDIC was
performed during uniaxial tensile loading. Dogbone-shaped samples were deformed inside of an SEM
and paused at various points during loading to collect highly magnified images of the sample surface. Two types of samples, one coated with OPS and one coated with gold speckles, were tested to examine
the strain partitioning at two length scales. The strain maps are shown in figure 5. HRDIC confirmed
that, as expected from the XRD, strain was accommodated first within the martensite regions. Additionally, the images show that the strain is not evenly distributed within the martensite regions. Rather, strong strain concentrations form along the interfaces between the α lamellae and martensite
regions (figure 5-a) and within the martensite regions themselves (figure 5-b). 4. Ti-6Al-4V Thus, in figure 4-d, a more negative value of the lattice strain implies that the sample takes
more load along the loading direction. 6 40th Risø International Symposium on Material Science ø
y p
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
g
doi:10.1088/1757-899X/580/1/012010 5. Strain accommodation in commercial superelastic NiTi 5. Strain accommodation in commercial superelastic NiTi
Superelasticity of NiTi allows it to sustain large strain without introducing permanent deformation
owing to the reversible martensitic transformation. The B2 austenite phase transforms into the B19’
martensite phase upon loading. Once the external load is removed, martensite can transform back to
austenite. The transformation behavior in the commercial superelastic nanocrystalline NiTi (Memry
GmbH) was studied by combining cruciform multiaxial deformation tests with in situ synchrotron X-
ray diffraction. Details about the sample geometries and experimental setups are given in reference [15]. y
p
g
p
p
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[
]
Figure 6 shows the evolution of the diffraction reflections in a selected 2θ range during a “square”
load path change. The initial microstructure is full austenite phase, as evidenced by the presence of
reflections only corresponding to the B2 phase before loading. During uniaxial loading along the F1
direction, austenite transforms into martensite when a critical transformation force (~24 N) is reached. The intensity of {110}B2 drops drastically, while the martensite reflections {002}B19’ and {012}B19’
appear. Subsequent loading along the F2 direction does not introduce new martensite reflections but
shows a slight decrease in the B19’ volume fraction, suggesting that a reverse transformation is induced
by changing the load path. Upon unloading along the F1 direction (changing loading condition from
F1F2 to F2max), the martensite reflections formed at F1max, {002}B19’ and {012}B19’, decrease in
intensity and eventually disappear. Meanwhile new martensite reflections, {110)B19’ and {020}B19’,
appear and grow in intensity. Different sets of martensite reflections corresponding to different loading
directions suggest a dependency of the martensitic variant selection on the load path, which is also
reported in [29], where uniaxial and equibiaxial loads result in different martensite footprints in
diffraction spectra. The final unloading of F2 induces a complete reverse transformation from martensite
to austenite. Upon unloading, the strong {110}B2 appears again, and no martensite reflection is retained. In situ X-ray diffraction clearly demonstrates the path dependency of the martensite variants during the
load path change in the commercial NiTi. Figure 6. Evolution of the diffraction intensity of different reflections during a square load path
change in NiTi alloy. Figure 6. Evolution of the diffraction intensity of different reflections during a square load path
change in NiTi alloy. As characterized by EBSD, the initial austenite microstructure is complex (see figure 7-a). IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
doi:10.1088/1757-899X/580/1/012010 The strain concentrations
between the martensite regions and α lamellae form very early in the deformation and imply that the α
lamellae are delaminating due to the mechanical differences between them. Delamination explains how
Phase-3 could shed load without deforming plastically in the microplastic regime. After microplastic
yield, strain concentrations are observed at the interfaces of the larger laths within the martensite regions
themselves (figure 5-b). This confirms the composite behavior of the martensite regions, which, prior to
these experiments, were thought to be homogeneous. Strain concentrations at the interfaces grow and
form microcracks, which ultimately lead to fracture of the samples. The in situ mechanical results imply 7 40th Risø International Symposium on Material Science 40th Risø International Symposium on Material Science
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 that varying the post-treatment annealing temperature can tune the volume fraction of the martensite
regions and the size of the laths within them to optimize work hardening and ductility. that varying the post-treatment annealing temperature can tune the volume fraction of the martensite
regions and the size of the laths within them to optimize work hardening and ductility. 5. Strain accommodation in commercial superelastic NiTi The large
austenite grains delineated with dashed line in figure 7-a contain bands varying in width, direction and
crystallographic orientation. High-magnification EBSD and TEM reveal that these bands consist of
agglomerations of subgrains (average crystallite size: 40 nm) having similar crystallographic
orientations. In order to correlate the transformation behavior observed by in situ X-ray diffraction with 8 40th Risø International Symposium on Material Science IOP Publishing y p
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
g
doi:10.1088/1757-899X/580/1/012010 f. Series: Materials Science and Engineering 580 (2019) 012010
doi:10.1088/1757-899X/580/1/012010 IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
doi:10.1088/1757-899X/5 the complex microstructure, in situ HRDIC using gold speckle pattern applied on the surface of the
cruciform-shape sample is performed. More experimental details are given in [15]. the complex microstructure, in situ HRDIC using gold speckle pattern applied on the surface of the
cruciform-shape sample is performed. More experimental details are given in [15]. Figure 7-b shows the in-plane strain maps at F1max and F2max during the square load path. Note
that the field of view for HRDIC is the same as the EBSD map shown in figure 7-a. HRDIC shows a
non-uniform strain distribution inside the large austenite grains, suggesting that the strain
accommodated by the phase transformation is microscopically heterogeneous. Depending on the loading
direction, the high-strain traces appear in different places in the same large austenite grains. For instance,
in Grain A, different inclinations and locations of the high-strain traces can be observed under F1max
and F2max. The high-strain traces mostly orient similarly to the band structure shown in the EBSD
crystallographic orientation map, underlying the importance of the initial austenite microstructure on
the transformation behavior. A careful inspection of the behavior in different grains confirms the
important role of the austenite subgrain structure [15]. Together with the in situ diffraction data shown
in figure 6, the link between the austenite microstructure and the transformation behavior during the
square load path change is uncovered. The martensitic transformation occurs in the nanoscaled austenite
subgrains. Depending on the loading direction, different groups of subgrains transform collectively into
different sets of martensitic variants, hence producing the heterogeneous strain distributions observed
in the HRDIC maps. After one cycle of the square load path change, diffraction data shows a full austenite phase and
slight peak broadening in austenite reflections, which implies the formation of dislocations. 5. Strain accommodation in commercial superelastic NiTi The HRDIC
strain map taken after unloading (figure 7-c) shows residual strain traces, which correspond to the sharp
contrast features in electron channeling contrast image (ECCI). These results affirm the formation of
dislocations during a single load path change cycle. Figure 7. (a) The initial microstructure of the commercial NiTi. A full B2 austenite phase is indexed
with EBSD. The subgrains are revealed in a higher magnification map. (b) The HRDIC strain map
taken at F1max and F2max during the square load path change. (c) The HRDIC strain map and the
electron channeling contrast image taken at the same area, after fully unloading. Figure 7. (a) The initial microstructure of the commercial NiTi. A full B2 austenite phase is indexed
with EBSD. The subgrains are revealed in a higher magnification map. (b) The HRDIC strain map
taken at F1max and F2max during the square load path change. (c) The HRDIC strain map and the
electron channeling contrast image taken at the same area, after fully unloading. References [1]
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[2]
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IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
IOP Publishing
doi:10.1088/1757-899X/580/1/012010 y p
IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
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doi:10.1088/1757-899X/580/1/012010 OP Conf. Series: Materials Science and Engineering 580 (2019) 012010
doi:10.1088/1757-899X/580/1/0 the Ti-6Al-4V alloy processed by EBM, in situ XRD revealed a rather complex load sharing between
three mechanically distinct phases, whereas HRDIC demonstrated an inhomogeneous strain
accommodation and delamination between very fine laths. Together the results of the two techniques
explain why this composite microstructure exhibits a high work hardening with good ductility. Finally,
synchrotron XRD showed that in a commercial NiTi alloy different martensite variants appear during a
strain path change, while HRDIC revealed how this strain is accommodated and underlined the role of
the nano-sized subgrain structure on the mechanical behavior of these material. Acknowledgements g
The authors thank the European Research Council for the ERC advanced Grant MULTIAX (339245). 6. Summary y
Three examples have been presented to highlight the complementary aspects of HRDIC and in situ
diffraction methods for investigating the mechanical behavior of engineering materials. Neutron
diffraction showed that in a 304 austenitic steel the martensitic transformation is enhanced during
equibiaxial deformation. HRDIC provided the additional understanding that under biaxial loading more
grains exhibit multiple slip systems, and hence more nucleation sites for martensite are provided. For Three examples have been presented to highlight the complementary aspects of HRDIC and in situ
diffraction methods for investigating the mechanical behavior of engineering materials. Neutron
diffraction showed that in a 304 austenitic steel the martensitic transformation is enhanced during
equibiaxial deformation. HRDIC provided the additional understanding that under biaxial loading more
grains exhibit multiple slip systems, and hence more nucleation sites for martensite are provided. For 9 40th Risø International Symposium on Material Science References 57 905–20 [13] Van Petegem S, Guitton A, Dupraz M, Bollhalder A, Sofinowski K, Upadhyay M V and Van
Swygenhoven H 2017 A Miniaturized Biaxial Deformation Rig for in Situ Mechanical Testing
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path change on superelastic NiTi alloys: In situ synchrotron XRD and SEM DIC Acta Mater. 144
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2016 In-situ neutron diffraction during biaxial deformation Acta Mater. 105 404–16 10 40th Risø International Symposium on Material Science IOP Conf. Series: Materials Science and Engineering 580 (2019) 012010
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A P and Li X 2007 Development of Patterns for Digital Image Correlation Measurements at
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[20] Polatidis E, Hsu W-N, Šmíd M and Van Swygenhoven H 2019 A High Resolution Digital
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2018 Suppressed martensitic transformation under biaxial loading in low stacking fault energy
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[24] Martin S, Wolf S, Martin U, Krüger L and Rafaja D 2016 Deformation Mechanisms in 4] Martin S, Wolf S, Martin U, Krüger L and Rafaja D 2016 Deformation Mechanisms in
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Effects of biodynamic preparations on the growth and yield parameters of potatoes with coloured flesh
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Biuletyn Instytutu Hodowli i Aklimatyzacji Roślin
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BIULETYN INSTYTUTU HODOWLI I AKLIMATYZACJI ROŚLIN ELVYRA JARIENĖ 1
HONORATA DANILČENKO 1
NIJOLE VAITKEVIČIENĖ 1
AGNIESZKA KITA 2
1 Aleksandras Stulginskis University, Agronomy Faculty, Institute of Agriculture and Food Sciences,
LT-53361 Kaunas district, Lithuania
2 Wroclaw University of Environmental and Life Sciences, Faculty of Food Science, Poland DOI: 10.37317/biul-2014-0032 DOI: 10.37317/biul-2014-0032 ∗ Redaktor prowadzący: Katarzyna Mikołajczyk Key words: biodynamic preparations, coloured fleshed potato, growth and yield parameters Rolnictwo biodynamiczne jest jedną z metod rolnictwa ekologicznego. W uprawie biodynamicznej,
w celu poprawy jakości plonu i kompozycji składników odżywczych w surowcu, wykorzystuje się
osiem konkretnych preparatów (z nawozu krowiego, krzemionki i różnych roślin). Materiałem użytym
do badań były ziemniaki odmian o kolorowym miąższu uprawiane w gospodarstwie ekologicznym
(powiat Prienai) w latach 2012–2013. Celem badań było określenie wpływu preparatów
biodynamicznych (BD) 500 i 501 na parametry wzrostu i wydajność plonu ziemniaków o kolorowym
miąższu. Eksperyment obejmował dwa czynniki: I — odmiany ziemniaka (dwie odmiany ziemniaków
z niebieskim miąższem — Blue Congo i Vitelotte oraz jedna z czerwonym — Red Emmalie), II —
wykorzystanie preparatów biodynamicznych do oprysków gleby i roślin (cztery zabiegi: 1. kontrola
bez preparatów BD 2. z zastosowaniem preparatu biodynamicznego 500; 3. z zastosowaniem preparatu
biodynamicznego 501; 4. z zastosowaniem obu preparatów biodynamicznych (500 + 501). Produktywność roślin ziemniaka oznaczano w doświadczeniu polowym (wartość indeksu zawartości
chlorofilu w liściach, masę i liczbę bulw pod krzewem). Najlepsze parametry wzrostu uzyskano przy
zastosowaniu kombinacji preparatów biodynamicznych (500 + 501). Stwierdzono, że w porównaniu z
wariantem kontrolnym stosowanie obu preparatów (500 + 501), istotnie zwiększyło zawartość
chlorofilu w liściach oraz masę i liczbę bulw z jednej rośliny w odmianach ziemniaka Blue Congo i
Red Emmalie. Preparat 501 istotnie zwiększał wskaźnik zawartości chlorofilu w liściach odmiany Red
Emmalie w 9 (1.07 razy) i 13 (1.13 razy) tygodniu, a Blue Congo tylko w 9 (1.07 razy) tygodniu po
posadzeniu. Niezbędne są jednakże dalsze badania, które pozwolą na ustalenie czy preparaty
biodynamiczne istotnie wpływają na parametry wzrostu i plonowanie bulw ziemniaka o kolorowym
miąższu. Słowa kluczowe: parametry wzrostu i plonu, preparaty biodynamiczne, ziemniak o kolorowym
miąższu Słowa kluczowe: Wpływ preparatów biodynamicznych na wzrost i plonowanie bulw ziemniaków
o kolorowym miąższu
Komunikat y
ą
Komunikat Biodynamic agriculture is one of the organic agricultural farming methods. Different from organic
farmers, biodynamic farmers add eight specific preparations (made from cow manure, silica, and
various plants) to improve crops growth and nutrient composition. In 2012–2013, in organic farm
(Prienai district), potato cultivars with coloured flesh were grown for research. The aim of two years’
research was to evaluate effects of biodynamic (BD) preparations (500 and 501) on the growth and
yield parameters of the coloured fleshed potatoes. An experiment included two factors: I — three potato
cultivars (purple fleshed — Vitelotte, Blue Congo and red-fleshed Red Emmalie), II — using of BD
preparations in field sprays (four treatments: 1. Control without BD preparations; 2. BD preparation
500; 3. BD preparation 501; 4. complex application of BD preparations (500+501). The individual
productivities of potato plants were analyzed in the field experiment (chlorophyll content index values
of leaves, tuber weight per plant and tuber number per plant). The results revealed that combination of
BD preparations (500 + 501) was the best among all the treatments for most of the growth and yield
parameters under study. It was found, that, compared with the control variant, combination of BD
preparations (500 + 501) substantially increased the chlorophyll content index in leaves, the weight and
number of tubers per plant of cvs. Blue Congo and Red Emmalie. BD preparation 501 substantially
increased the chlorophyll content index in leaves of Red Emmalie at 9th (1.07 times) and 13th (1.13
times) week, and of Blue Congo only at 9th (1.07 times) week after planting. However, more research
is needed to determine whether the BD preparations affect growth and yield parameters of colour-
fleshed potato tubers. 73 73 Elvyra Jarienė ... Słowa kluczowe: parametry wzrostu i plonu, preparaty biodynamiczne, ziemniak o kolorowym
miąższu INTRODUCTION Coloured potatoes (Solanum tuberosum L.) have attracted the attention of researchers
as well as purchasers because of their antioxidant activities, appearance and taste. The
antioxidant activity in these potatoes is related to the presence of anthocyanins, flavonoids,
ascorbic acid, tocopherols, alpha-lipoic acid and selenium. Consequently, coloured
potatoes have the potential to be one of the most significant sources of antioxidants in the
human diet (Lachman et al., 2005; Nayak et al., 2011; Jarienė et al., 2013). In order to
preserve the nutritive value of these potatoes, one needs to look for alternative farming
methods. Like organic farming, the products of biodynamic agriculture are nutritionally superior
and they taste better than the conventional foods, besides having the potential to mitigate
some of the negative effects of chemical agriculture (Steiner, 1996). Biodynamic agriculture was developed in the 1920 s in response to concerns from
farmers about the deteriorating soils and health of their farms (Steiner, 1993). Similar to
organic agriculture, biodynamics eliminates synthetic chemical fertilizers and pesticides. A major difference is that biodynamic farmers add eight specific preparations to their soils,
crops, and composts to enhance soil and crop quality and to stimulate the composting
process (Zaller and Kopke, 2004). In order to promote efficient cycling of nutrients, a series
of eight fermented herbal and manure based preparations numbered 500 through 507 are
applied regularly, in small quantities to the soil, compost and crops. Some biodynamic 74 Elvyra Jarienė ... farmers make the preparations themselves while others buy them from certified biodynamic
associations or experienced practitioners (Reganold, 1995; Koepf et al., 2001). The preparation 500 consists of high quality farmyard manure, fresh or aged, put in
bovine horns, then buried at autumn and dug up in spring, after that it can be stored under
controlled conditions for some months and finally sprayed to the soil (Koepf et al., 2001). y p y
(
p
)
Horn silica (501) is powdered quartz (rock crystal) put in a bovine horn and processed
as horn manure. A very small quantity of the 501 is then dissolved in water and sprayed on
the standing crop, mostly at flowering stage: it would reinforce the plant against pests and
diseases and improve its nutritional properties, flavours and shelf-life (Koepf et al., 2001;
Catellani, 2006). INTRODUCTION These two BD preparations are believed to work synergistically, with
preparation 500 mainly improving the overall soil fertility, and preparation 501 being active
in enhancing the plant physiological response to the light radiation (Spaccini et al., 2012). Biodynamic preparations are added to the soil or to composting organic material always
in very low doses of a few grams per ton of soil/compost material. Therefore, the primary
purpose of these compounds is not to add nutrients, but to stimulate the processes of
nutrient and energy cycling, to affect decomposition/building of humus and to improve soil
and crop quality (Raupp, 1999). The main aim of this study was to establish effects of biodynamic (BD) preparations
(500 and 501) on the growth and yield parameters of the colour-fleshed potatoes. MATERIALS AND METHODS Field experiment was carried out in 2012-2013 in organic farm (Prienai district). When
applying biodynamic (BD) preparations, potatoes were grown using the traditional potato
growing technology (Ražukas, 2003). They were planted in May, and harvested in
September. The field experiment was carried out in four replications. Variants of
replications were arranged randomly. The overall field size of potato experiment was 17.5
m2 whereas the size of the accounting field was 10 m2. Agrochemical soil indicators were following: weakly acidic (pHKCl 6.86), phosphorus
(166.1 mg·kg-1), very high in potassium (248.8 mg·kg- 1) and low in nitrogen (0.142%). An experiment of two factors was carried out: I – three potato cultivars with coloured
tuber flesh (Vitelotte and Blue Congo — blue and Red Emmalie — red), II — biodynamic
(BD) preparations — 500 and 501 — used for potato field spraying. There were four
treatments to evaluate the effectiveness of BD preparations: 1. control (BD preparations
were not used); 2. application of BD preparation 500 (the soil was sprayed two weeks
before the planting of potatoes with 1% solution); 3. application of BD preparation 501
(two times early in the morning potato leaves were sprayed with 0.5% solution in the VIII
and IX stages of organogenesis); 4. complex application of BD preparations (500+501)
(two weeks before the planting of potatoes the soil was sprayed with 1% solution of BD
preparation 500 and two times early in the morning potato leaves were sprayed with 0.5%
solution of BD preparation 501 in the VIII and IX stages of organogenesis). BD preparations (500 and 501) investigated in the experiment, were purchased in the
Biodynamic Preparations Centre, Germany. During experiment it was evaluated values of 75 Elvyra Jarienė ... chlorophyll content index (CCI) in leaves of potatoes (chlorophyll content meter CCM 200
Plius, Opti-Science, Tyngsboro, MA, USA) — in the 9th and 13th week after the planting. Ten plants were randomly selected in each replication. The CCM-200 plus uses absorbance
to estimate the chlorophyll content in leaf tissue. Two wavelengths were used for
absorbance determinations. One wavelength falls within the chlorophyll absorbance range
while the other one serves to compensate for mechanical differences such as tissue
thickness. The device measures the absorbance of both wavelengths and calculates a
chlorophyll content index (CCI) value that is proportional to the amount of chlorophyll in
the sample. RESULTS AND DISCUSSION Values of chlorophyll content index (CCI) are directly proportional to the total content
of chlorophyll in leaves. According to the CCI of the foliage, it is possible to predict the
yield and to accurately determine its relationship with index values recorded during various
stages of growth (Spaner et al., 2005; Gianquinto, 2004; Zebarth et al., 2007; Janušauskaitė,
2009). Results of our research have shown that CCI values in potato leaves depend on the
cultivar, BD preparations used and age of plants (Table 1). The highest values of CCI were
found in the leaves of cv. Red Emmalie 9 and 13 weeks after planting of potatoes. In all
the measurements the lowest CCI values were determined in cv. Vitelotte leaves. It is stated
in the literature that the pigment content may be influenced by architectonics of the plant’s
leaves, leaning angle towards the sun which varies in different varieties (Long et al., 2006). Our research results have shown that in comparison with control, complex using of BD
preparations (500 + 501) substantially increased CCI values in cvs. Blue Congo and Red
Emmalie leaves 9 (respectively 1.10 and 1.08 times) and 13 (respectively 1.25 and 1.16
times) weeks after planting whereas the use of preparations in cv. Vitelotte had no
significant effect. Comparing with control, BD preparation 501 substantially increased the
CCI values in cv. Red Emmalie leaves after 9 (1.07 times) and 13 (1.13 times) weeks
whereas in Blue Congo only 9 (1.07 times) weeks after potato planting (Table 1). Tegethoff
and Koepf (1993) also found that horn-silica (BD preparations 501) spray increased
chlorophyll content in bush beans. BD preparation 500 did not affect CCI values in the
leaves of grown cultivars. Values of chlorophyll content index (CCI) are directly proportional to the total content
of chlorophyll in leaves. According to the CCI of the foliage, it is possible to predict the
yield and to accurately determine its relationship with index values recorded during various
stages of growth (Spaner et al., 2005; Gianquinto, 2004; Zebarth et al., 2007; Janušauskaitė,
2009). Results of our research have shown that CCI values in potato leaves depend on the
cultivar, BD preparations used and age of plants (Table 1). The highest values of CCI were
found in the leaves of cv. Red Emmalie 9 and 13 weeks after planting of potatoes. In all
the measurements the lowest CCI values were determined in cv. Vitelotte leaves. MATERIALS AND METHODS The average tuber mass per plant, expressed in g·plant-1 and tuber number per
plant were also assessed. Soil analyses were conducted at the Laboratory of Food Raw Materials, Agronomic and
Zootechnical Research of Aleksandras Stulginskis University. Soil pHKCl was established
by the potentiometric method in 1N KCl extract. The amount of total nitrogen in the soil
was established by the Kjeldahl method. The amount of available phosphorus was
determined by the CAL method using a spectrophotometer as well available potassium
content using a flame photometer. Statistical analysis was performed using two-way ANOVA (STATISTICA software). Statistical significance was considered at p<0.05. Arithmetic means and standard
deviations of research data were calculated with EXCEL program. RESULTS AND DISCUSSION It is stated
in the literature that the pigment content may be influenced by architectonics of the plant’s
leaves, leaning angle towards the sun which varies in different varieties (Long et al., 2006). Our research results have shown that in comparison with control, complex using of BD
preparations (500 + 501) substantially increased CCI values in cvs. Blue Congo and Red
Emmalie leaves 9 (respectively 1.10 and 1.08 times) and 13 (respectively 1.25 and 1.16
times) weeks after planting whereas the use of preparations in cv. Vitelotte had no
significant effect. Comparing with control, BD preparation 501 substantially increased the
CCI values in cv. Red Emmalie leaves after 9 (1.07 times) and 13 (1.13 times) weeks
whereas in Blue Congo only 9 (1.07 times) weeks after potato planting (Table 1). Tegethoff
and Koepf (1993) also found that horn-silica (BD preparations 501) spray increased
chlorophyll content in bush beans. BD preparation 500 did not affect CCI values in the
leaves of grown cultivars. 76 Elvyra Jarienė ... Plant age also had a significant influence on the CCI values. Essentially negative effect
on CCI values was established after 13 weeks of potato planting (Table 1). Poljak and
others (2008) determined that CCI values significantly decreased when plant got older. RESULTS AND DISCUSSION Table 1
Effects of biodynamic (BD) preparations on chlorophyll content index values (CCI) of potato plants
during growing season
Wpływ preparatów biodynamicznych na wartości indeksu zawartości chlorofilu (WIK) w liściach
roślin ziemniaka podczas wegetacji
Treatment
Doświadczenie
Chlorophyll content index values (CCI)
Wartość zawartości chlorofilu (CCI)
9 weeks after planting
9 tygodni po posadzeniu
13 weeks after planting
13 tygodni po posadzeniu
Blue Congo
Control without BD preparations
20.46 ± 0.97 b
8.63 ± 1.17 b
BD preparation 500
20.68 ± 1.37 ab
9.70 ± 0.81 b
BD preparation 501
21.99 ± 1.93 a
9.36 ± 0.75 b
BD preparations 500 + 501
22.47 ± 1.91 a
10.80 ± 0.77 a
Vitelotte
Control without BD preparations
14.93 ± 1.05 a
6.76 ± 0,90 a
BD preparation 500
14.15 ± 1.94 a
7.13 ± 0.80 a
BD preparation 501
15.80 ± 0.86 a
7.88 ± 1.03 a
BD preparations 500 + 501
15.91 ± 1.34 a
7.32 ± 1.07 a
Red Emmalie
Control without BD preparations
32.49 ± 1.91 b
19.45 ± 1.38 b
BD preparation 500
32.40 ± 1.71 b
19.43 ± 1.96 b
BD preparation 501
34.92 ± 1.87 a
22.10 ± 1.15 a
BD preparations 500+ 501
34.99 ± 1.99 a
22.50 ± 1.59 a
*— Means located on the same column and marked with different letters differ significantly at p<0.05
* — Wyniki znajdujące się w tej samej kolumnie i oznaczone różnymi literami różnią się istotnie, gdy p <0,05 Table 1
Effects of biodynamic (BD) preparations on chlorophyll content index values (CCI) of potato plants
during growing season Potato yield is determined by the number and weight of tubers per plant (Čeponienė,
2002). The largest number and weight of tubers per plant were determined in cv. Red
Emmalie whereas the smallest — in cv. Blue Congo (Table 2). It was established that in comparison with control, only the complex of BD preparations
(500+501) essentially increased the yield of potato tubers. Average number of tubers per
plant in cv. Red Emmalie and Blue Congo increased by 1.12 and 1.13 times respectively,
as well average weight of tubers per plant increased by 1.10 and 1.34 times respectively. However, separately used BD preparations 500 and 501 had no influence on tuber yield in
these cultivars (Table 2). The use of BD preparations did not affect the yield of tubers of Vitelotte cultivar either,
there were no essential differences among the treatments. RESULTS AND DISCUSSION There are other authors who
mention different effect of BD preparation on various potato cultivars (Granstedt and
Kjellenberg, 1997). 77 Elvyra Jarienė ... Table 2
Effects of biodynamic (BD) preparations on the yield parameters of potato tubers
Wpływ preparatów biodynamicznych na parametry plonu bulw ziemniaków
Treatment
Doświadczenie
Number of tubers per plant
Liczba bulw na roślinę
Tuber mass per plant (g)
Ciężar bulw na roślinę (g)
Blue Congo
Control without BD preparations
8.25 ± 0.27 b
371.25 ± 17,5 b
BD preparation 500
8.31 ± 0.42 b
365.13 ± 22.17 b
BD preparation 501
8.35 ± 0.37 b
377.53 ± 27.95 b
BD preparations 500 + 501
9.30 ± 0.65 a
496.24 ± 34.97 a
Vitelotte
Control without BD preparations
15.99 ± 0.43 a
473,22 ± 21.17 a
BD preparation 500
16.03 ± 0.56 a
451.95 ± 38.67 a
BD preparation 501
16.15 ± 0.41 a
460,42 ± 35.28 a
BD preparations 500 + 501
16.46 ± 0.44 a
472.95 ± 56.19 a
Red Emmalie
Control without BD preparations
16,48 ± 0.55 b
1045.13 ± 25.90 b
BD preparation 500
16.98 ± 0.60 b
1037.50 ± 43.49 b
BD preparation 501
17.30 ± 0.30 b
1085.45 ± 34.16 ab
BD preparations 500 + 501
18.41 ± 0.41 a
1116.59 ± 42.81 a
*— Means located on the same column and marked with different letters differ significantly at p<0.05
* — Wyniki znajdujące się w tej samej kolumnie i oznaczone różnymi literami różnią się istotnie, gdy p <0,05 Effects of biodynamic (BD) preparations on the yield parameters of potato tubers
Wpływ preparatów biodynamicznych na parametry plonu bulw ziemniaków y
Gianquinto, G., Goffart, J.P., Olivier, M. et al. 2004. The use of hand-held chlorophyll meters as a tool to assess
the nitrogen status and to guide nitrogen fertilization of potato crop. Potato Research. 47, Nr. 5: 35 — 80. CONCLUSIONS The results revealed that combination of BD preparations (500 + 501) was the best
among all the treatments for most of the growth and yield parameters under study. It was found, that compared with the control variant, combination of BD preparations
(500 + 501) substantially increased the chlorophyll content index values in leaves of cvs. Red Emmalie and Blue Congo in 9th (respectively 1.10 and 1.08 times) and 13th
(respectively 1.25 and 1.16 times) week after planting. BD preparation 501 substantially
increased the chlorophyll content index in leaves of cv. Red Emmalie in 9th (1.07 times)
and 13th (1.13 times) week, and of cv. 'Blue Congo' only in 9th (1.07 times) week after
planting. Combination of BD preparations (500 + 501), compared with the control variant,
substantially increased the weight and number of tubers per plant of cvs. Blue Congo and
Red Emmalie. BD preparations did not affect the growth and yield parameters of cv. Vitelotte. BD preparations did not affect the growth and yield parameters of cv. Vitelotte. p
Čeponienė S. 2002. Nematodams atsparių labai ankstyvų ir ankstyvų bulvių veislių tyrimas. Sodininkystė ir
daržininkystė. LSDI ir LŽŪU mokslo darbai. Nr. 21 (1): 72 —77. LITERATURE Catellani, G. 2006. Guida all’Agricoltura Biodinamica. Associazione per l’Agricoltura Biodinamica, Milano,
p. 142. p
Čeponienė S. 2002. Nematodams atsparių labai ankstyvų ir ankstyvų bulvių veislių tyrimas. Sodininkystė ir
daržininkystė. LSDI ir LŽŪU mokslo darbai. Nr. 21 (1): 72 —77. 78 Elvyra Jarienė ... Granstedt A., Kjellenberg L. 1997. Long-Term Field Experiment in Sweden: Effects of Organic and Inorganic
Fertilizers on Soil Fertility and Crop Quality. In Proceedings of an International Conference in Boston,
Tufts University, Agricultural Production and Nutrition, Massachusetts March 19–21, 1997. y
g
Janušauskaitė, D. 2009. Trešimo intensyvumo įtaka vasarinių kvietrugių produktyvumui bei chlorofilo indeksui
vasarinių kvietrugių lapijoje. Agriculture. T. 96, Nr. 4: 110 — 123. ų
g ų
p j j
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Jarienė E., Vaitkevičienė N., Chupakhina N., Poltavskaya R. L., Kita A. 2013. Antioxidant compounds and
antioxidant activity in blue-fleshed potatoes. Rural development 2013: The 6th international scientific
Conference Proceedings, ASU: 119 — 122. g ,
Koepf H. H., Schaumann W., Haccius M. 2001. Agricoltura Biodinamica. Antroposofica Editrice, p. 366. Lachman J., Hamouz K., Orsak M. 2005. Red and purple potatoes — A significant antioxidant source in human
nutrition. Chem. Lists. 99: 474 — 482. Long S., Zhu X. G., Naidu S., Ort D. 2006. Can improvement in photosynthesis increase crop yields? Plant,
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antioxidant-rich value-added foods. J. of Food Proc. and Preser. 35 (5): 571 — 580. Poljak, M., Horvat, T., Majic, A., Pospisil, A., Cosic, T. 2008. Nitrogen management for potatoes using rapid
test methods. Alps-adria Scientific Workshop. Cereal Research Comm. 36: 1795 — 1798. Raupp, J. 1999. Biodynamic approaches in research and development. In Research Methodologies in Organic
Farming. R. Zanoli and R. Krell (Eds.), REU Technical Series 58, FiBL, FAO, Rome. g
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Ražukas A. 2003. Bulvės. Biologija, selekcija, sėklininkystė. Vilnius. Ražukas A. 2003. Bulvės. Biologija, selekcija, sėklininkystė. Vilnius. Reganold, J. P. 1995. Soil quality and profitability of biodynamic and conventional farming systems: A review. Am. J. of Alternative Agriculture. 10: 36 — 45. Richardson A. D., Duigan S. P., Berlyn G. P. 2002. An evaluation of noninvasive methods to estimate foliar
chlorophyll content. New Phytologist. 153: 185 — 194. Spaccini R., Mazzei P., Squartini A., Giannattasio M., and Piccolo A. 2012. LITERATURE Molecular properties of a fermented
manure preparation used as a field spray in biodynamic agriculture. Envirom. Sci. and Pollution R. 19:
4214 — 4225. Spaner D., Todd A. G., Navabi A. McKenzie D. B., Goonewardene L. A. 2005. Can leaf chlorophyll measure
at differing growth stages be used as an indicator of winter wheat and spring barley nitrogen requirements
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Hudson, NY. Steiner R. 1996. Spiritual Foundations for the Renewal of Agriculture: A course of lectures. Biodynamic
Farming and Gardening Association, Kimberton, p. 310. Tegethoff M., In: Koepf H. H. 1993. Research in biodynamic agriculture: methods and results. Bio-Dynamic
Farming and Gardening Association Inc., Kimberton, p. 48. g
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Zaller J. G., Köpke U. 2004. Effects of traditional and biodynamic farmyard manure amendment on yields, soil
chemical, biochemical and biological properties in a long-term field experiment. Biology and Fertility of
Soils, 40: 222 — 229. Zebarth, B. J., Botha E. J. Rees H. 2007. Rate and time of fertilizer nitrogen application on yield, protein and
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718. 79 79
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https://openalex.org/W3033456202
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https://www.frontiersin.org/articles/10.3389/fneur.2020.00476/pdf
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English
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An Experimental Investigation of White Matter Venous Hemodynamics: Basic Physiology and Disruption in Neuroinflammatory Disease
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Frontiers in neurology
| 2,020
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cc-by
| 7,246
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ORIGINAL RESEARCH
published: 02 June 2020
doi: 10.3389/fneur.2020.00476 An Experimental Investigation of
White Matter Venous Hemodynamics:
Basic Physiology and Disruption in
Neuroinflammatory Disease
Scott C. Kolbe 1*, Sanuji. I. Gajamange 2, Jon O. Cleary 3 and Trevor J. Kilpatrick 4
1 Department of Neuroscience, Central Clinical School, Prahran, VIC, Australia, 2 Walt and Eliza Hall Institute, Parkville, VIC,
Australia, 3 Department of Radiology, Guy’s and St. Thomas’ NHS Foundation Trust, London, United Kingdom, 4 Florey
Institute of Neuroscience and Mental Health, Parkville, VIC, Australia The white matter is highly vascularised by the cerebral venous system. In this paper,
we describe a unique blood oxygen-level dependent (BOLD) signal within the white
matter using functional MRI and spatial independent components analysis. The signal is
characterized by a narrow peak frequency band between 0.05 and 0.1 Hz. Hypercapnia,
induced transient increases in white matter venous BOLD that disrupted the oscillation
indicative of a vasocontractile mechanism. Comparison of the white matter venous BOLD
oscillations between 14 healthy subjects and 18 people with perivenular inflammation
due to multiple sclerosis (MS), revealed loss of power in the white matter venous
BOLD signal in the peak frequency band (patients = 6.70 ± 0.94 dB/Hz vs. controls
= 7.64 ± 0.71 dB/Hz; p = 0.006). In MS, lower power was associated with greater
levels of neuroinflammatory activity (R = −0.64, p = 0.006). Using a signal modeling
technique, we assessed the anatomical distribution of white matter venous BOLD signal
abnormalities and detected reduced power in the periventricular white matter, a region
of known venous damage in MS. These results demonstrate a novel link between
neuroinflammation and vascular physiological dysfunction in the cerebral white matter,
and could indicate enduring loss of vascular compliance associated with imperfect repair
of blood-brain barrier damage after resolution of acute neuroinflammation. Edited by:
Achim Gass,
University Medical Center
Mannheim, Germany
Reviewed by:
Yann Quidé,
School of Psychiatry, UNSW, Australia
Volker Rasche,
University of Ulm, Germany
*Correspondence:
Scott C. Kolbe
scott.kolbe@monash.edu Edited by:
Achim Gass,
University Medical Center
Mannheim, Germany Reviewed by:
Yann Quidé,
School of Psychiatry, UNSW, Australia
Volker Rasche,
University of Ulm, Germany ,
School of Psychiatry, UNSW, Australia
Volker Rasche, *Correspondence:
Scott C. Kolbe
scott.kolbe@monash.edu Specialty section:
This article was submitted to
Applied Neuroimaging,
a section of the journal
Frontiers in Neurology INTRODUCTION Received: 11 February 2020
Accepted: 01 May 2020
Published: 02 June 2020 Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system. The
initiating neuroinflammatory events of multiple sclerosis remain unclear, however, the pathological
hallmark of the disease involves peripheral immune cell infiltration via the internal cerebral venous
system of the white matter (1) upon disruption to the blood-brain-barrier, leading to perivenular
demyelination and axonal injury. Imaging studies have shown that a majority of MS patients have
perivenular lesions, leading to recent proposals to include the so-called “central vein sign” as an aid
for differential diagnosis (2–4). Keywords: white matter, cerebral venous system, hemodynamics, neuroinflammation, cerebral veins, multiple
sclerosis BOLD MRI Pre-processing and Spatial
Independent Components Analysis p
p
y
BOLD-weighted MRI scans were processed using FSL MELODIC
spatial-ICA software (18). The processing pipeline was as follows
for each subject. (1) BOLD time-series image data were linearly
realigned to correct for head motion and gradient heating,
and registered to the single-band image. One patient and one
control subject were excluded due to severe head motion (>1 mm
inter-scan motion) during BOLD imaging. (2) Data were high
pass filtered (pass band > 0.01 Hz) to remove low frequency
signal drift due to MRI gradient heating and spatially smoothed
using a non-linear edge-detection based algorithm (19) with
spatial extent of 5 mm. (3) Spatial ICA was performed on
each subject. Subjects Eighteen participants [7 m/9f; mean (SD) age at the time of
testing = 42.4 (10.3) yrs] were recruited prospectively between
2008 and 2010 upon presenting with first demyelinating event
acute optic neuritis at the Royal Victorian Eye and Ear Hospital as
part of a published trial investigating optic nerve imaging during
and after acute optic neuritis (13). Scanning for the present study
was performed between 3 and 5 years after initial recruitment as
part of an extension study. At the time of scanning, all patients
had been diagnosed with clinically definite multiple sclerosis
based on the 2010 revisions to the McDonald criteria (14). Fourteen control subjects [6 m/9f; mean (SD) age at the time
of testing = 32.5 (4.6) yrs] were also recruited. This study was
approved by the Royal Victorian Eye and Ear Hospital and Royal
Melbourne Hospital Human Research Ethics Committees. All
participants provided voluntary written consent in accordance
with the Declaration of Helsinki. The resulting independent component maps were manually
inspected and the white matter component was identified
for each subject based on two anatomical features: (a)
within the white matter, and (b) in close proximity to
the lateral ventricles. Potential differences between patients
and controls in the spectral qualities of the independent
component time-courses (peak power and frequency) were
tested for using Student’s t-tests. Pearson’s correlation analyses
were used to test for co-variation between peak power
and frequency. Citation: Kolbe SC, Gajamange SI, Cleary JO
and Kilpatrick TJ (2020) An
Experimental Investigation of White
Matter Venous Hemodynamics: Basic
Physiology and Disruption in
Neuroinflammatory Disease. Front. Neurol. 11:476. doi: 10.3389/fneur.2020.00476 Despite the relevance of the white matter venous system to multiple sclerosis pathology,
relatively few studies have directly studied venous pathology in the multiple sclerosis June 2020 | Volume 11 | Article 476 Frontiers in Neurology | www.frontiersin.org White Matter Hemodynamics Kolbe et al. brain. Pathological studies have reported enduring damage to
the venous system in multiple sclerosis after the resolution of
acute inflammation (5, 6). Neuroimaging studies have reported
reduced density (7–9), caliber (10, 11), and perfusion (12) of
the white matter venous system in multiple sclerosis patients. Together, these findings indicate that the influx of peripheral
inflammatory cells into the brain via the cerebral venous system
leads to enduring anatomical and physiological changes to the
vessels. It is conceivable that such pathologies could confer a
susceptibility to further acute inflammation or lead to hypo-
perfusion, thus exacerbating neuronal injury. in-plane acceleration (2x acceleration). In addition, a single-band
image was acquired for each subject (no multi-band acceleration
but all other parameters same) which, because of its enhanced
gray/white matter contrast, was used for registrations between
echo planar imaging and T1 anatomical space. Lesions were delineated on the double inversion recovery
images
using
a
semi-automated
thresholding
technique
(16). Intra-cranial, whole brain parenchyma, gray matter
and white matter volumes were obtained using Freesurfer
(version
5.0.4)
and
the
standard
analysis
pipeline
(17). Freesurfer segmentations were all assessed by the author
and subsequently used to calculate brain, gray matter and white
matter volumes, normalized to the intra-cranial volume for
each subject. To further investigate physiological changes in the cerebral
venous system, we assessed the blood oxygen-level dependent
(BOLD) signal in the white matter using resting-state functional
MRI. We show that the cerebral venous system is characterized by
a unique oscillatory BOLD signal that is disrupted in people with
early multiple sclerosis, and that the degree of signal disruption
is associated with the degree of neuroinflammation. Frontiers in Neurology | www.frontiersin.org MRI Acquisitions All MRI scans were performed using a 3 Tesla MRI system
(Trio TIM, Siemens, Erlangen, Germany) with a 32-channel
receiver head coil. Three MRI sequences were acquired for each
subject: (1) A 3D whole brain double inversion recovery sequence
for lesion identification (repetition/echo/inversion times =
7,400/324/450 and 3,000 ms; flip angle = 120◦; resolution 0.55
× 0.55 × 1.1 mm3 sagittal acquisition); (2) A 3D whole brain
MPRAGE T1-weighted sequence for volumetric assessments
(repetition/echo/inversion time = 1900/2.63/900 ms; flip angle =
9◦; resolution 0.8 × 0.8 × 0.8 mm3 sagittal acquisition); and (3)
A BOLD-weighted echo planar imaging sequence acquired while
subjects watched a blank screen with eyes open (repetition/echo
times = 1,400/33 ms; flip angle = 85◦; resolution 2.5 × 2.5
× 2.5 mm3 axial acquisition; number of BOLD measurements
= 440). High spatial and temporal resolution was obtained
through the use of “multi-band” simultaneous multi-slice echo
planar imaging acquisition (3x acceleration) (15) and GRAPPA Independent component maps were transformed to standard
MNI-152 brain space using a three stage non-linear registration
procedure utilizing the Advanced Normalization Tools (ANTs)
software (20). (1) Each subject’s single-band echo planar imaging
reference image was non-linearly registered to the subject’s T1
anatomical image. (2) Each subject’s T1 anatomical image was
registered to the MNI-152 T1 standard brain. (3) The two
deformation fields from steps (1) and (2) were added together
and the independent component maps were transformed to MNI
space using the resulting field. Example venous independent
component maps in standard MNI space are shown for two
patients and two control subjects in Figure 1A. Potential
differences in the anatomical distribution of the IC maps between
patients and controls was tested using voxel-wise general linear
models and FSL’s RANDOMIZE non-parametric permutation
sampling statistical tool (21). Resulting statistical maps were June 2020 | Volume 11 | Article 476 Frontiers in Neurology | www.frontiersin.org 2 Kolbe et al. White Matter Hemodynamics FIGURE 1 | Visual flow chart of the processing pipeline for generating the white matter venous BOLD map. (A) Raw resting-state BOLD times series data was
preprocessed to correct for head motion, spatially and temporally filtered and then input to spatial ICA decomposition. (B) All IC component maps were visualized and
the white matter venous component identified based on it’s location and power spectral features oscillatory signal with peak power ∼0.05 Hz. MRI Acquisitions (C) For each subject,
the white matter venous component was non-linearly aligned to MNI space, thresholded at Z > 2.3 and binarized. (D) All subjects’ binarised maps were averaged to
create a standard space voxelwise probability map which was used to sample resting-state signals from all subjects for group analyses. FIGURE 1 | Visual flow chart of the processing pipeline for generating the white matter venous BOLD map. (A) Raw resting-state BOLD times series data was
preprocessed to correct for head motion, spatially and temporally filtered and then input to spatial ICA decomposition. (B) All IC component maps were visualized and
the white matter venous component identified based on it’s location and power spectral features oscillatory signal with peak power ∼0.05 Hz. (C) For each subject,
the white matter venous component was non-linearly aligned to MNI space, thresholded at Z > 2.3 and binarized. (D) All subjects’ binarised maps were averaged to
create a standard space voxelwise probability map which was used to sample resting-state signals from all subjects for group analyses. prior to downloading according to the protocol described in
Glasser et al. (24) including motion correction and nonlinear
registration to MNI-152 atlas space. After downloading we
performed spatially smoothed using a non-linear edge-detection
based algorithm (19) with spatial extent threshold of 4 mm, and
temporally high pass filtered (pass band > 0.01 Hz) prior to
single-subject ICA for each dataset. Spatial ICA was performed
using FSL MELODIC. The analysis was constrained to obtain
only 80 components for each subject to limit the processing time. This number was found to be liberal enough to reliably identify
the white matter component. The white matter component was
identified in each subject by an experienced observer (SK) in all
but three subjects. Data for these three subjects was observed to
be high in temporal noise and multi-band artifacts (between slice
signal correlations). corrected for multiple comparisons using the threshold-free
cluster enhancement algorithm (22). To generate a probabilistic standard space map of the
white matter veins based on the independent component maps,
each subject’s standard space independent component map was
thresholded (Z > 2.3) and binarised. The resulting binary masks
were averaged across all subjects to obtain a map where each
voxel value represented the proportion of subjects where that
voxel was within the venous independent component map. The
entire processing pipeline is summarized in Figure 1. Replication Using Human Connectome
Project Data To confirm the spatial and spectral features of the white matter
venous BOLD signal, we replicated the previous analyses in an
independent dataset of 30 healthy resting state fMRI datasets
obtained from the Human Connectome Project (HCP) (23). Thirty random healthy resting-state 3 Tesla fMRI datasets
were obtained from the Washington University—University
of Minnesota Human Connectome Project (23). Prior to
downloading, the datasets had been minimally pre-processed RESULTS Characterization of the White Matter
Venous BOLD Signal in Healthy Subjects
In all subjects a spatial ICA component was identifiable in
the periventricular white matter (Figure 2A). Single sample
statistical analysis demonstrated a large region after family
wise error correction across most of the cerebral white matter
(Figure 2B). Characterization of the White Matter
Venous BOLD Signal in Healthy Subjects
In all subjects a spatial ICA component was identifiable in
the periventricular white matter (Figure 2A). Single sample
statistical analysis demonstrated a large region after family
wise error correction across most of the cerebral white matter
(Figure 2B). g
To test the effects of non-neurally driven BOLD signal changes
on the white matter venous system, three healthy male subjects
(ages = 39/36/31) underwent two runs of functional MRI
whilst performing a simple breath hold task to induce transient
hypercapnia. The first run consisted of 7 repetitions of 18 s breath
hold followed by 24 s normal breathing. The second run consisted
of 24 s of breath hold followed by 18 s of normal breathing. BOLD functional MRI data were pre-processed to correct for
motion, high-pass and spatially filtered as previously described
above. Mixed-effects general linear models were used to test for
the following main effects: condition (hypercapnia vs. baseline),
tissue type (gray vs. white matter), and duration (short vs. long
breath hold), and following interactions: condition/tissue type
and condition/tissue type/duration. Post-hoc correlation analyses
were used to compare BOLD signal changes between tissue types. g
y
j
In all subjects a spatial ICA component was identifiable in
the periventricular white matter (Figure 2A). Single sample
statistical analysis demonstrated a large region after family
wise error correction across most of the cerebral white matter
(Figure 2B). The anatomy of the signal was investigated more precisely in a
single subject using high-resolution functional and susceptibility-
weighted
MRI
at
7
Tesla
(Supplementary Figure 1). Susceptibility weighted imaging reveals the venous blood
vessels within the white matter as regions of high magnetic
susceptibility due to the presence of blood. The ICA component
was most evident in regions characterized by medium to large
periventricular veins. Power spectral features of the white matter venous BOLD
time-series were also assessed. In all subjects, white matter
venous BOLD signals were characterized by a narrow peak
frequency band was observed in the range of 0.05–0.070 Hz
[mean (SD) = 0.061 (0.008) Hz; peak power (SD) = 34.87
(7.20) dB/Hz]. White Matter Venous BOLD Voxel Wise
Group Comparisons neurological or vascular disease) was imaged using a 7 Tesla
MRI System (Magentom, Siemens, Erlangen). Two sets of images
were collected: (1) a gradient echo sequence for calculation
of the susceptibility weighted image (repetition/echo times =
24/17.34 ms; flip angle = 13◦; resolution 0.75 × 0.75 × 0.75 mm3
axial acquisition); and (2) a multi-band BOLD-weighted echo
planar imaging sequence with imaging parameters comparable
to those used for 3T MRI (repetition/echo times = 1,500/25 ms;
flip angle = 44◦; resolution = 2 × 2 × 2 mm3 axial acquisition;
number of BOLD measurements = 205), acquired while subjects
watched a blank screen with eyes open. A data driven signal modeling approach was used to test for
putative anatomical differences in the white matter venous BOLD
signal between patients and controls. For each brain voxel,
high-pass filtered BOLD signals (<0.01 Hz) were converted
to power spectra using multi-taper spectral decomposition in
MATLAB R⃝R2016a. Each voxel’s power spectrum was fitted
with a Gaussian curve using nonlinear least squares with
appropriate boundary conditions (0.04<µ<0.1 Hz; σ
>0 Hz)
using MATLAB R⃝R2016a. The fitted power and frequency
for the spectral peak was compared voxelwise between groups
using a general linear model permutation sampling method
(RANDOMIZE, FSL, FMRIB, Oxford, UK) (21) family-wise error
corrected with threshold-free cluster enhancement (22). BOLD data were processed using MELODIC according to the
methods described above for 3 Tesla data and linearly registered
to the high-resolution gradient echo image using FLIRT (FSL,
FMRIB, Oxford, UK). 7TESLA MRI VENOGRAPHY AND BOLD
IMAGING To confirm the venous origin of the white matter IC map, a
single healthy volunteer (26 year old female with no history of June 2020 | Volume 11 | Article 476 Frontiers in Neurology | www.frontiersin.org 3 Kolbe et al. White Matter Hemodynamics RESULTS Peak power and frequency were not correlated
(R = −0.28). White Matter Venous BOLD Spectral
Analysis In order to compare the power spectra of actual BOLD signal
time-courses between patients and controls, rather than IC
time-courses, the white matter probability map was used to
calculate the weighted-average power-spectrum for all brain
voxels for each subject. Briefly, raw BOLD time-course data
were pre-processed to correct for head motion and perform high
pass filtering. No spatial smoothing was performed. For each
voxel, the BOLD time-course was converted to a power spectrum
using non-parametric multi-taper spectral decomposition (25)
implemented in the MATLAB R⃝R2016a Signal Processing
Toolbox
(mathworks.com/help/signal/ref/pmtm.html). A
weighted mean power spectrum was calculated for each subject
using the weighting values from the spatial probability map. The average power within a conservatively judged peak region
(0.04 to 0.1 Hz) was compared between patients and control
subjects using a Student’s t-test. Pearson’s correlation analyses
were performed between average power and logarithmically
transformed
lesion
volume
and
brain,
gray,
and
white
matter fractions. Replication Analysis of the White Matter
Venous BOLD Signal Using HCP Dataset g
g
Compared to the mean white matter IC map for our dataset, the
HCP dataset displayed a lower average magnitude (Figure 3A). The same narrow peak frequency band was observed in the
HCP data and did not differ significantly in peak frequency or
power from our dataset [mean frequency (SD) = 0.057 (0.008)
Hz, Student’s t (33 degrees of freedom) = 1.36, p = 0.20; mean
peak power (SD) = 32.09 (6.12) dB/Hz, t33 = 1.33, p = 0.21]
(Figure 3B). There was no correlation between peak power and
frequency (R = −0.23). The white matter component was undetectable in three
HCP subjects. To explore this further we compared temporal
SNR between our data and the HCP data to determine
whether temporal noise could reduce the detectability of the June 2020 | Volume 11 | Article 476 Frontiers in Neurology | www.frontiersin.org 4 Kolbe et al. White Matter Hemodynamics FIGURE 2 | Spatial maps of the white matter venous BOLD signal output from spatial independent components analysis. (A) Shows individual component maps for
the white matter venous BOLD signal. (B) Shows the regions of significant correspondence across subjects using a single group t-test, family-wise error corrected
using the threshold-free cluster enhancement (TFCE) method. FIGURE 2 | Spatial maps of the white matter venous BOLD signal output from spatial independent components analysis. (A) Shows individual component maps for
the white matter venous BOLD signal. (B) Shows the regions of significant correspondence across subjects using a single group t-test, family-wise error corrected
using the threshold-free cluster enhancement (TFCE) method. FIGURE 2 | Spatial maps of the white matter venous BOLD signal output from spatial independent components analysis. (A) Shows individual component maps for
the white matter venous BOLD signal. (B) Shows the regions of significant correspondence across subjects using a single group t-test, family-wise error corrected
using the threshold-free cluster enhancement (TFCE) method. = 2.5 mm isotropic, HCP = 2 mm isotropic) and higher
multiband acceleration factor (our data = 3, HCP = 5) used
by HCP. However, despite noisier and thus less reliable data,
the HCP data recapitulated the gross spatial and temporal
features of the white matter venous BOLD signal observed in
our data. venous component. DISCUSSION This study represents, to the best of our knowledge, the first
in-depth study of the hemodynamics of the white matter using
BOLD-weighted MRI. The white matter venous BOLD signal
has been reported previously in the context of removal of
artifactual signals from resting state neural connectivity analysis
[see Figure 3 from Salimi-Khorshidi et al. (26)]. Perivenular
lesions are a consistent feature of multiple sclerosis (27), and
the high degree of spatial congruence between the white matter
venous BOLD signal and regions of high lesion load in multiple
sclerosis (28) led us to explore the signal further as a potential
marker for venous pathophysiology. We confirmed that the
white matter venous BOLD signal co-localized with smaller and
larger internal cerebral venous system using high resolution
susceptibility-weighted imaging at 7 Tesla. In our experience, Replication Analysis of the White Matter
Venous BOLD Signal Using HCP Dataset We observed a relatively large decrease
in temporal SNR in the HCP data compared to our data
∼60%, despite the large number of extra fMRI volumes
collected for each subject (n = 400 for our data compared
to n = 1,200 for HCP data). This difference was likely
due to a combination of smaller voxel dimensions (our data June 2020 | Volume 11 | Article 476 Frontiers in Neurology | www.frontiersin.org 5 Kolbe et al. White Matter Hemodynamics FIGURE 3 | Comparison of spatial and spectral characteristics for the white matter venous BOLD signal between this study the Human Connectome Project (HCP). (A) Shows the high degree of spatial congruence between the mean component maps of the two data sets. (B) The spectral properties of the white matter venous
BOLD signal were similar between the datasets, with both showing a narrow peak high power band between 0.04 and 0.1 Hz. FIGURE 3 | Comparison of spatial and spectral characteristics for the white matter venous BOLD signal between this study the Human Connectome Project (HCP). (A) Shows the high degree of spatial congruence between the mean component maps of the two data sets. (B) The spectral properties of the white matter venous
BOLD signal were similar between the datasets, with both showing a narrow peak high power band between 0.04 and 0.1 Hz. BOLD Power Spectrum Modeling BOLD Power Spectrum Modeling
In healthy subjects, peak power was constricted to the white
matter (Figure 5A upper) demonstrating the specificity of
the spectral characteristics to the white matter. Similarly, the
multiple sclerosis group showed peak power constrained
to
the
white
matter,
yet
the
magnitude
of
the
power
was diminished compared
to control (Figure 5A lower). Voxel-wise statistical analyses revealed significant loss of
power
in
the
periventricular
white
matter
(Figure 5B),
consistent with the location of multitudinous small veins
that are a common site of inflammatory demyelination in
multiple sclerosis. White Matter Venous BOLD Physiology
During Hypercapnia volume (R = −0.65, p = 0.005; Figure 4E), but not with non-
inflammatory markers of brain injury including normalized
brain (R = 0.22), gray matter (R = 0.31), or white matter
volumes (R = 0.33). These overall statistical results were not
affected by narrowing the frequency band used for calculating
mean power. Firstly, we observed a significant main effect of condition
[F(1948,1) = 53.8, p < 0.0001) demonstrating a clear effect
of hypercapnia on the BOLD signal. Secondly, we detected
a significant interaction between condition and tissue type
[F(1948,1) = 1269, p < 0.0001] demonstrating that gray matter
and white matter responded differently to hypercapnia. post-hoc
comparisons of BOLD change between gray and white matter
showed that while gray matter BOLD reduced as expected
(1 marginal means = −1.50, p < 0.0001), white matter
venous BOLD signals significantly increased (1 marginal
means = 0.98, p < 0.0001). The degree of BOLD increase
in the white matter was negatively correlated with gray
matter BOLD decreases in all three subjects for both short
(subject 1: R = −0.44, p < 0.0001; subject 2: R = −0.73,
p < 0.0001; subject 3: R = −0.50, p < 0.0001) and long
(subject 1: R = −0.35, p < 0.0001; subject 2: R = −0.63,
p < 0.0001; subject 3: R = −0.46, p < 0.0001) breath hold runs
(Supplementary Figure 2). White Matter Venous BOLD Alterations in
Early Multiple Sclerosis A standard space white matter venous map (Figure 4A) was
used to calculate the weighted average raw BOLD signal time-
courses for the white matter for each subject (voxels within
T2 lesions in patients were omitted in patients). Average white
matter venous BOLD power across a conservatively judged
peak frequency range (0.04–0.1 Hz) was significantly lower in
multiple sclerosis patients (6.70 ± 0.94 dB/Hz, Figure 4C)
compared to control subjects [7.64 ± 0.71 dB/Hz, t(28) =
2.9, p = 0.002, Figure 4B] when compared using a t-test
(Figure 4D). Variation in the mean power in this band in patients
correlated significantly with logarithmically transformed lesion June 2020 | Volume 11 | Article 476 Frontiers in Neurology | www.frontiersin.org 6 Kolbe et al. White Matter Hemodynamics GURE 4 | Comparison of the white matter venous BOLD signal between healthy and multiple sclerosis participants. (A) The probabilistic map of the BOLD signal
ross all subjects. Values (0–1) reflect the proportion of subjects with the component in each voxel. Maximal congruence was observed in regions corresponding to
e location of the peri-ventricular veins and small veins within the corona radiata. These are also the most common sites of neuro-inflammatory activity in multiple
erosis. (B,C) Weighted average power spectra from within the probability map for control and multiple sclerosis subjects demonstrate a clear peak power within the
04–0.1 Hz frequency band. (D) Mean power within this band was significantly reduced in multiple sclerosis cases compared to control subjects (** = p < 0.01), and
mean power in patients significantly correlated with logarithmically transformed cerebral lesion volume. FIGURE 4 | Comparison of the white matter venous BOLD signal between healthy and multiple sclerosis participants. (A) The probabilistic map of the BOLD signal
across all subjects. Values (0–1) reflect the proportion of subjects with the component in each voxel. Maximal congruence was observed in regions corresponding to
the location of the peri-ventricular veins and small veins within the corona radiata. These are also the most common sites of neuro-inflammatory activity in multiple
sclerosis. (B,C) Weighted average power spectra from within the probability map for control and multiple sclerosis subjects demonstrate a clear peak power within the
0.04–0.1 Hz frequency band. (D) Mean power within this band was significantly reduced in multiple sclerosis cases compared to control subjects (** = p < 0.01), and
(E) mean power in patients significantly correlated with logarithmically transformed cerebral lesion volume. White Matter Venous BOLD Alterations in
Early Multiple Sclerosis white matter venous BOLD signals are almost always observed
in single subject ICA output but are generally not analyzed. Our analyses confirmed that the white matter venous BOLD
signal is highly stereotypic across individuals in terms of anatomy
and spectral properties. We were also able to confirm spatial
and temporal features of white matter venous BOLD in a
completely independent dataset (HCP). We expect that the signal could be obtained from any resting-state fMRI data
given the reasonably low frequency of the signal (∼0.05 Hz)
compared to the acquisition frequencies used in most fMRI
studies (0.33–1.3 Hz). To better understand the physiological mechanisms driving
the white matter venous BOLD oscillation, we employed dynamic
hypercapnic challenge (breath holding) during fMRI scanning June 2020 | Volume 11 | Article 476 Frontiers in Neurology | www.frontiersin.org 7 Kolbe et al. White Matter Hemodynamics FIGURE 5 | Comparisons of modeled BOLD power between multiple sclerosis and control subjects. (A) Average modeled peak power of the BOLD signal for control
and multiple sclerosis subjects illustrates a reduction in peak power (dB/Hz) across the much of the white matter. (B) The t-statistic map shows regions of significant
reduction in peak power in the multiple sclerosis group compared to control subjects after correction using threshold-free cluster enhancement. FIGURE 5 | Comparisons of modeled BOLD power between multiple sclerosis and control subjects. (A) Average modeled peak power of the BOLD signal for control
and multiple sclerosis subjects illustrates a reduction in peak power (dB/Hz) across the much of the white matter. (B) The t-statistic map shows regions of significant
reduction in peak power in the multiple sclerosis group compared to control subjects after correction using threshold-free cluster enhancement. likely to reflect cyclic vasodilation and constriction under central
sympathetic control. Further studies will be required confirm
the sympathetic origin of the signal and to elucidate the local
control mechanisms. in three healthy subjects. Hypercapnia is a potent vasodilator in
cerebral gray matter, causing marked BOLD signal attenuation in
the cortex commensurate with increased partial volume inclusion
of low contrast blood. We therefore expected that hypercapnia
induced vasodilation would also affect the white matter venous
BOLD signal. In contrast to cortical BOLD, hypercapnia caused
a transient increase in the white matter venous BOLD signal the
strength of which temporally correlated with the degree of BOLD
decrease in cortex. White Matter Venous BOLD Alterations in
Early Multiple Sclerosis A likely cause for this increase is a reduction
in the voxel partial volume inclusion of low contrast venules
and veins associated with vasoconstriction. Vasoconstriction in
the white matter venous system under hypercapnia could be
a mechanism for maintaining intracranial pressure and blood
volume during the large vasodilatory response in gray matter
regions characteristic of hypercapnia. This is consistent with the
well-recognized role of veins as blood capacitance vessels (29). In multiple sclerosis patients, power in the white matter
venous BOLD signal was reduced commensurate with the
degree of neuroinflammatory lesion load. This suggests that the
physiological substrates of BOLD signal power loss in people
with multiple sclerosis relates to inflammatory damage. The
white matter venules and veins are the principal entry point
for peripheral immune cells into the brain through a disrupted
blood-brain barrier during acute inflammation. Such damage to
the vascular wall acutely could lead to ongoing postacute damage
or imperfect repair. Indeed, previous studies have reported
venous atrophy (7–9) and altered blood flow (12) in the white
matter of people with multiple sclerosis. Three studies have
also reported reduced venous density in multiple sclerosis (7–9),
most likely associated with venous atrophy to the point where
the blood vessels can no longer be visualized, even with the
high resolution (<=0.5 × 0.5 mm2 in-plane) afforded by 7
Tesla MRI (8). Consistent with our findings of a link between
neuroinflammation and venous damage, Sinnecker et al. (8)
showed that venous density was negatively correlated with T2
lesion load. Venous atrophy is a potential substrate for loss of
BOLD signal power via the reduction of partial volume inclusion
of the venous signal in imaging voxels. Also potentially associated
with venous atrophy, reduced venous flow has been reported
in the periventricular white matter in both clinically isolated
syndromes and relapsing-remitting multiple sclerosis patients
using dynamic contrast MRI (12). The authors also observed a
non-significant trend toward reduced blood volume, suggestive
of vascular atrophy. Gaitán et al. (11) reported narrowing of
veins within T2 lesions, yet enlargement of perilesional veins g
p
At rest, the white matter venous BOLD signal was observed to
oscillate at around one cycle every 20 s. This rate excludes cardiac
and respiratory influences and instead points to a potential auto-
regulatory function. Cerebral blood flow is tightly regulated and
is resistant to rapid changes in systemic arterial blood pressure
(30). Frontiers in Neurology | www.frontiersin.org ACKNOWLEDGMENTS We sincerely thank all study participants for their time. We acknowledge the Traditional Owners of the land on
which this research was conducted and pay respects to
their Elders, past and present. We acknowledge the financial
support for the research provided by the National Health
and Medical Research Council (APP10009757, APP1054147),
National Multiple Sclerosis Society (RG4211A4/2). Data were
provided (in part) by the Human Connectome Project, WU-
Minn Consortium (Principal Investigators: David Van Essen
and Kamil Ugurbil; 1U54MH091657) funded by the 16 NIH
Institutes and Centers that support the NIH Blueprint for
Neuroscience Research; and by the McDonnell Center for
Systems Neuroscience at Washington University. A version of
this manuscript has been released as a pre-print at bioRxiv
(Kolbe et al. https://doi.org/10.1101/208751). ETHICS STATEMENT The studies involving human participants were reviewed
and approved by Royal Victorian Eye and Ear Hospital
Human Research Ethics Committee Royal Melbourne Hospital
Human Research Ethics Committee. The patients/participants
provided their written informed consent to participate in
this study. y
This study has several limitations that should be addressed
in follow-up studies. Firstly, we did not directly anatomically
image and map the venous system in all subjects, so it was not
possible to compare the haemodynamic alterations to venous
anatomy and morphology directly. The high resolution afforded
by high field (7T+) MRI allows the mapping of fMRI signals to
specific blood vessels, and to create maps of the white matter
venous system that could be used to better characterize the spatial
distribution of venous damage. We did however, exclude voxels
from each patient’s white matter venous probability map that
were classified as lesion on double inversion recovery scans. Therefore, the changes in white matter venous BOLD power
observed in patients was measured from normal appearing
brain regions that were not influenced by overt signal changes
associated with lesion pathology. Finally, our study focused
on a convenience sample of patients with a history of acute
optic neuritis with a relatively consistent and short disease
duration of between 3 and 5 years. Future studies should
characterize the progression of white matter haemodynamic
abnormalities in later disease stages. Longitudinally designed
studies will also be required to determine whether white
matter haemodynamic abnormalities are associated with greater
susceptibility to subsequent inflammatory cell infiltration or
neurodegenerative changes. DATA AVAILABILITY STATEMENT Raw anonymised data will be made available upon request to
the authors. White Matter Venous BOLD Alterations in
Early Multiple Sclerosis The observed BOLD frequency range is within the limits
of both myogenic (0.02–0.15 Hz) and sympathetic (0.07–0.15 Hz)
vascular regulation (31). In support of a myogenic mechanism,
mechanoreceptors have been identified in the walls of the cerebral
venous system that are activated by an increase in cerebral blood
volume to regulate blood flow (32). The degree of correlation of
myogenic regulation across the venous system is unknown. In
contrast, sympathetic activity is centrally controlled and known
to control vasoconstriction within the cerebral venous system
(33). Given the high degree of signal coordination across the
entire internal cerebral venous system in healthy individuals,
we hypothesize that the observed oscillatory BOLD signal is June 2020 | Volume 11 | Article 476 8 Kolbe et al. White Matter Hemodynamics compared to healthy control veins using ultra-high resolution
T2∗-weighted MRI (0.5 × 0.5 × 0.5 mm3) during gadolinium
infusion. The authors interpreted their findings to indicate that
perivascular inflammation could lead to vascular compression
and thickening of the perivascular wall. Supporting evidence
can be found in histological studies of the vascular system in
multiple sclerosis noting fibrinoid and haemosiderin deposition,
thrombosis, and venous wall thickening (5, 6). Given the
large capacitance of the venous system, it is possible that the
perilesional enlargement observed by Gaitán et al. (11) reflects a
bottleneck effect caused by flow resistance within lesions, leading
to perilesional vascular distension. Together, these studies
demonstrate significant neuroinflammatory damage to the white
matter venous system that could account for the haemodynamic
changes observed in our study. It is conceivable that reduced
hemodynamics has the potential to exacerbate neural damage via
local ischemia. This hypothesis support further investigation of
the white matter venous BOLD signal in the context of other
diseases characterized by white matter lesions. the white matter veins that initiates early in the disease. Future studies are required to identify the physiological
mechanism driving white matter venous BOLD hemodynamics
and explore the role of altered hemodynamics in multiple
sclerosis pathophysiology. SUMMARY AND CONCLUSIONS The internal cerebral veins within the white matter are the most
common site of early peripheral immune cell infiltration in
the neuroinflammatory demyelinating disease multiple sclerosis. This study identified a novel haemodynamic signal with a
narrow spectral peak in the cerebral veins of the white
matter using resting-state functional MRI and ICA. Peak
power of the signal was reduced in people with multiple
sclerosis, and the degree of reduction correlated significantly
with neuroinflammatory lesion volume. These results indicate
that multiple sclerosis is associated with dysfunction of AUTHOR CONTRIBUTIONS SK conceived of the study, collected and analyzed data
and
wrote
the
manuscript. SG
made
contributions
to
the interpretation of data for the work and revised the
paper critically for important intellectual content. JC made
contributions to the interpretation of data for the work
and revised the paper critically for important intellectual
content. TK made contributions to the interpretation of data
for the work and revised the paper critically for important
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online
at:
https://www.frontiersin.org/articles/10.3389/fneur. 2020.00476/full#supplementary-material June 2020 | Volume 11 | Article 476 9 White Matter Hemodynamics Kolbe et al. REFERENCES White matter hemodynamic abnormalities precede sub-cortical gray
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absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. 15. Xu J, Moeller S, Auerbach EJ, Strupp J, Smith SM, Feinberg DA, et al. Evaluation of slice accelerations using multiband echo planar imaging at 3 T. Neuroimage. (2013) 83:991–1001. doi: 10.1016/j.neuroimage.2013.07.055 Copyright © 2020 Kolbe, Gajamange, Cleary and Kilpatrick. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
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Clinical and genetic characterization of chanarin-dorfman syndrome patients: first report of large deletions in the ABHD5 gene
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Orphanet journal of rare diseases
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cc-by
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Open Access Open Access Abstract Background: Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive disorder characterized by
nonbullous congenital ichthyosiform erythroderma (NCIE) and an intracellular accumulation of triacylglycerol (TG)
droplets in most tissues. The clinical phenotype involves multiple organs and systems, including liver, eyes, ears,
skeletal muscle and central nervous system (CNS). Mutations in ABHD5/CGI58 gene are associated with CDS. Methods: Eight CDS patients belonging to six different families from Mediterranean countries were enrolled for
genetic study. Molecular analysis of the ABHD5 gene included the sequencing of the 7 coding exons and of the
putative 5’ regulatory regions, as well as reverse transcript-polymerase chain reaction analysis and sequencing of
normal and aberrant ABHD5 cDNAs. Results: Five different mutations were identified, four of which were novel, including two splice-site mutations
(c.47+1G>A and c.960+5G>A) and two large deletions (c.898_*320del and c.662-1330_773+46del). All the reported
mutations are predicted to be pathogenic because they lead to an early stop codon or a frameshift producing a
premature termination of translation. While nonsense, missense, frameshift and splice-site mutations have been
identified in CDS patients, large genomic deletions have not previously been described. Conclusions: These results emphasize the need for an efficient approach for genomic deletion screening to ensure
an accurate molecular diagnosis of CDS. Moreover, in spite of intensive molecular screening, no mutations were
identified in one patient with a confirmed clinical diagnosis of CDS, appointing to genetic heterogeneity of the
syndrome. feature of NLSD since birth, the disorder is referred to
as Chanarin-Dorfman syndrome (CDS [MIM 275630])
or neutral lipid storage disease with ichthyosis (NLSDI). Patients are sometimes born as collodion babies. Serum
lipids are normal, whereas muscle and hepatic enzymes
are frequently elevated compared to control values. Besides granulocytes and other blood cells, LDs are seen
in multiple cell types, including fibroblasts, basal kerati-
nocytes, myocytes. and hepatocytes. Severe hepatic stea-
tosis consequent to liver LD accumulation can cause life
threatening portal hypertension [7,8]. Although ichthyo-
sis is always present, other clinical features of CDS may
vary. The biochemical defect found in all the CDS
patients is attributed to deficient fatty acid (FA) mobili-
zation [9-12]. As a consequence of this impairment, a Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 © 2010 Redaelli et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Clinical and genetic characterization of
chanarin-dorfman syndrome patients: first report
of large deletions in the ABHD5 gene Chiara Redaelli1, Rosalind A Coleman2, Laura Moro3, Catherine Dacou-Voutetakis4, Solaf Mohamed Elsayed5,
Daniele Prati6,7, Agostino Colli8, Donatella Mela9, Roberto Colombo10, Daniela Tavian1* * Correspondence: daniela.tavian@unicatt.it
1Department of Psychology, Catholic University of the Sacred Heart, Milan,
Italy
Full list of author information is available at the end of the article Introduction Neutral-lipid storage diseases (NLSDs) are a clinically
heterogeneous group of non-lysosomal inherited disor-
ders characterized by the cytoplasmic accumulation of
lipid droplets (LDs) in most tissues. Clinical phenotypes
include myopathy (skeletal and heart muscle), liver
damage, ataxia, neurosensory hearing loss, ichthyosis,
sub-capsular cataracts, nystagmus, strabismus, and,
rarely, mental retardation [1-6]. When non-bullous con-
genital ichthyosiform erythroderma (NCIE), presenting
as fine scaling on erythematous skin, is the dominant y
ll list of author information is available at the end of the article Page 2 of 11 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 criteria were: congenital ichthyosiform erythroderma
characterized by fine scales on an erythematous back-
ground, hepatomegaly or liver steatosis, raised serum
levels of aminotransferases, and the presence of Jordans’
bodies in granulocytes. The involvement of other organs
and systems (spleen, eyes, ears, skeletal muscles, bone
marrow, and CNS) was variable. Complete clinical eva-
luation of each CDS patient has been reported elsewhere
(Table 1). Skin biopsies were obtained from three
patients and two relatives, and fibroblast cultures were
established in minimal essential medium with Earle’s
salts supplemented with 10% foetal bovine serum. The
cells were subcultured by trypsinization, as required
(5-15 passages), and aliquots were stored under liquid
nitrogen. When fibroblast cultures were not available,
DNA and RNA were extracted by conventional methods
from whole blood samples drawn from patients and
their relatives. number of tissues other than adipose accumulate TGs in
LDs even in the absence of an excess of circulating FAs. LDs even in the absence of an excess of circulating FAs. CDS is inherited as an autosomal recessive disorder
and has been reported in approximately 55 cases, parti-
cularly in families whose origins are in the Mediterra-
nean area and the Middle-East [13]. However, the
presence of CDS Families from Saudi Arabia, India and
Japan has also been reported [14]. The identification of
mutations in ABHD5 gene (originally called CGI58
[UniGene Hs.19385]), located on chromosome 3p21, in
nine CDS families from Algeria, Morocco, Turkey, and
France (13) strengthened the suggestion that CDS is a
unique clinical variety of NLSDs with a defined genetic
cause. The ABDH5 gene is comprised of 7 exons that
encompass about 28 kb of genomic DNA [MIM
604780]. Genomic analysis
Oli
l
id Oligonucleotides were selected to amplify and sequence
the seven exons of ABHD5, their intron/exon bound-
aries, and the candidate promoter regions. The primer
sequences are reported in Additional file 2 Table S1. PCR was performed in a 50 μl mixture containing 200
ng of genomic DNA, using a PTC-200 thermocycler (MJ
Research). PCR conditions for genomic amplifications of
exons 2-7 were reported by Lefèvre et al. [13] To
amplify exon 1, we used the DyNAzyme EXT (Finn-
zymes), and the PCR reaction was performed in 10%
DMSO (annealing: 50°C). Cell microscopy Peripheral blood smears or buffy coats were stained with
the May-Grünwald-Giemsa and Nile Red (NR) stain to
detect LDs in neutrophils and in monocytes by 100×
light and fluorescence microscopy [24]. Fibroblasts were
cultured on glass coverslips, allowed to adhere over-
night, observed under phase-contrast light microscopy
(40×; IX51, Olympus), fixed with 3.7% paraformaldeyde
and stained with NR prior to fluorescence microscopy
(40×). NR (Sigma-Aldrich) staining solution was freshly
prepared in DPBS (1:100 v/v) from a saturated solution
(1 mg/ml) in dimethylsulfoxide. Fluorescent images
were captured using a Leica MB5000B microscope
equipped with a DFC480 R2 digital camera and a Leica
Application Suite (LAS) software. To date, 22 point mutations and small insertions/dele-
tions have been identified in the ABHD5 gene. We now
report a molecular study of six additional CDS families
from Southern Italy, Egypt, Palestine and Greece. Genetic analysis of ABHD5 coding regions and their
flanking DNA sequences, as well as RT-PCR analysis of
ABHD5 complete cDNA, allowed us to identify novel
mutations, thus expanding the allelic spectrum of chro-
mosome-3p21-linked CDS to large genomic deletions. The failure to detect any functional ABHD5 genomic
variation in one family provides evidence for the genetic
heterogeneity of CDS. Introduction The cDNA has an open reading frame of 1427
nucleotides that predict a 349-amino acid protein of
approximately 39 kDa. ABDH5 has been reported to
have two functions, one as a cofactor for adipose trigly-
ceride lipase (ATGL) [15], and the other as a lysopho-
sphatidic acid acyltransferase [16,17]. ATGL is a TG
hydrolase that promotes the catabolism of stored fat in
adipose and non adipose tissues [15]. The ATGL gene
(alias PNPLA2) has been identified as the causative gene
for the neutral lipid storage disease with myopathy with-
out ichthyosis (NLSDM) [18]. Patients and methods
Families and Specimens Eight patients for whom a diagnosis of CDS had been
unambiguously established and eight unaffected relatives
from six families were investigated for ABHD5 muta-
tions (Table 1). Two families were from Italy (Molise:
patient A-II-1; Sicily: patient F-II-1) [7,19], one family
from Egypt (patient B-II-1) [20], one from Palestine
(patients C-II-2 and C-II-1) [21], one from Cyprus
(patient D-II-2) [22] and one from Greece (Athens:
patients E-II-1 and E-II-2) [23]. Two families were
known to be consanguineous because of marriages
between first cousins (Additional file 1 Figure S1). Signed, informed consent was obtained from each
patient and each family member. Common diagnostic The sequence spanning 5 kb upstream from the ATG
starting codon of the ABHD5 gene was scanned for
transcription factors AP-2, Sp1, GCF, NF-D, T-Ag by
the program PROSCAN Version 1.7. Two putative pro-
moter sequences were identified through this analysis;
they were localized at about 5 and 0.3 Kb upstream
from the ATG starting codon of the ABHD5 gene, Page 3 of 11 Page 3 of 11 Redaelli et al. Results
Patients Patients
The two main clinical features of CDS (i.e. NCIE and
hepatomegaly or liver steatosis) were present in all
families investigated in this study, but with considerable
variation in the extent and degree of organ involvement
in individual patients (Table 1). Hepatosplenomegaly
and steatosis were first observed in patient B-II-1 as
early as 9 months of age and in D-II-2, E-II-1 and E-II-
2 patients at age 20-22 months. In patients F-II-1 and
A-II-1, hepatosplenomegaly was noted at age 16 and 42,
respectively, when the patients were diagnosed clinically. Reduction in liver size was observed in patient E-II-1
(3 years old) after a medium-chain TG diet. At the age
of 8 years, still on the special diet, the E-II-1 patient had
a normal liver size and normal plasma aminotransferase
activities [23]. Serum aminotransferases were moderately
elevated in all patients, with the exception of C-II-2 e
C-II-1. Pediatric-age cataracts were present in patients
C-II-3, C-II-2, D-II-2, E-II-1 and E-II-2. On ophthalmo-
logic examination, bilateral nuclear cataracts were also
seen in A-II-1 at age 42. Bilateral ectropion was the
only ocular abnormality in patient B-II-1. No ophthal-
mologic abnormalities were observed for F-II-1. As
reported by other authors, clinical evidence of myopathy
in NCIE patients usually begins in their thirties. Never-
theless, as shown by abnormal electromyography, one of
our pediatric patients, B-II-1, presented with myopathy
and C-II-2, C-II-1 and D-II-2 patients (13, 12 and 9
years old, respectively) presented with a mild myopathy. Serum creatine kinase was elevated in C-II-2, C-II-1 and
D-II-2 patients, but was normal in B-II-1. Neurological
abnormalities are generally considered to be a late mani-
festation of the disease. Most of our patients did not
show any neurological impairment, with the exception
of C-II-2 and C-II-1 patients. In these two patients, pure
tone audiometry also demonstrated neurosensory
deafness p
p
ABHD5 large deletions were identified amplifying
genomic DNA with 6F/7R and 4aF/6R primer pairs. PCR conditions for 4aF/6R primers were as follows: hot
start at 96°C for 5 min; denaturation at 94°C for 40 sec,
annealing at 55°C for 40 sec, extension at 68°C for 5
min for 30 cycles; denaturation at 94°C for 40 sec,
annealing at 55°C for 40 sec and terminal extension at
68°C for 30 min for the last cycle. The JumpStart Accu-
Taq LA DNA Polymerase (Sigma) was used to perform
the long-range PCR of this large fragment. Patients and methods
Families and Specimens The aF/aR cycling profile was as fol-
lows: denaturation at 94°C for 4 min, annealing at 50°C
for 30 sec and extension at 70°C for 3 min for the first
round; denaturation at 94°C for 30 sec, annealing at 50°C
for 30 sec and extension at 70°C for 3 min for 30 cycles;
denaturation at 94°C for 30 sec, annealing at 50°C for 30
sec and terminal extension at 70°C for 10 min for the last
cycle. The PCR reaction was performed in 25 μl with 4%
of DMSO, using the DyNAzyme EXT. 0.5 μl of aF/aR
PCR was used to perform the next amplification with bF/
bR primers. 30 cycles of amplification were performed
using Taq Polymerase and the same cycling profile of aF/
aR primers with the exception of the extension (45 sec). spanning respectively 251 and 245 nt. The first promoter
region was amplified using 1F/1R primers, spanning from
-313 nt of the ABDH5 promoter to 338 nt of ABDH5
exon 1. A nested PCR was performed, using aF/aR and
bF/bR primer pairs, to analyze the second putative
promoter region. The aF/aR cycling profile was as fol-
lows: denaturation at 94°C for 4 min, annealing at 50°C
for 30 sec and extension at 70°C for 3 min for the first
round; denaturation at 94°C for 30 sec, annealing at 50°C
for 30 sec and extension at 70°C for 3 min for 30 cycles;
denaturation at 94°C for 30 sec, annealing at 50°C for 30
sec and terminal extension at 70°C for 10 min for the last
cycle. The PCR reaction was performed in 25 μl with 4%
of DMSO, using the DyNAzyme EXT. 0.5 μl of aF/aR
PCR was used to perform the next amplification with bF/
bR primers. 30 cycles of amplification were performed
using Taq Polymerase and the same cycling profile of aF/
aR primers with the exception of the extension (45 sec). turation for 5 min at 94°C, 37 cycles of denaturation for
30 sec at 94°C, annealing for 30 sec at 56°C, and exten-
sion for 1 min at 72°C. Specific sets of primers were
selected to reveal the consequences of ABHD5 nucleotide
variations localized in splice-site sequences; they were
2aF/2R for c.47+1G>A and 9aF/9R for c.960+5G>A
mutations. PCR conditions for 2aF/2R and 9aF/9R primer
pairs were the same as those used for 9F/9R. Patients and methods
Families and Specimens Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Table 1 Summary of Patients’ clinical data
Clinical features
Patients
A-II-1
B-II-1
C-II-2
C-II-1
D-II-2
E-II-1
E-II-2
F-II-1
Age/sex
42 y (F)
1 y (M)
12 y (M)
13 y (M)
9 y (M)
8 y (M)
6 y (M)
16 y M)
Place of origin
Molise
Egypt
Palestine
Palestine
Greece-Cyprio
Greece
Greece
Sicily
Consanguinity
No
Yes
Yes
Yes
No
No
No
No
Lipid vacuoles in
Granulocytes and
monocytes
Granulocytes, monocytes, skin,
liver, bone marrow, epidermal
Langerhans cells
Granulocytes,
monocytes, skin
Granulocytes,
monocytes,
skin
Granulocytes,
keratinocytes,
fibroblasts, endothelial
cells
Granulocytes,
monocytes, skin
Granulocytes,
monocytes, skin
Granulocytes,
monocytes, skin,
liver
Liver disease
Severe steatosis,
splenomegaly, portal
hypertension
Hepatosplenomegaly, steatosis
NE
NE
Hepatomegaly
Hepatomegaly,
fatty infiltration,
lobular fibrosis
Hepatomegaly,
fatty infiltration,
lobular fibrosis
Hepatomegaly
NCIE
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Myopathy
No
Yes
Mild
Mild
Mild
No
No
No
Ophtalmological
(Ophthalmologic
examination)
Cataracts
Bilateral ectropion
Cataracts
(Nuclear)
Cataracts
(Nuclear)
microcataracts, myopia
(Nuclear)
Cataracts
Cataracts
No
Deafness
Hypoacusia
No
Yes
Yes
No
No
No
No
CNS abnormalities
No
No
Neurological
retardation
Neurological
retardation
No
No
No
No
Altered
biochemical
analysis
AST, ALT
Triglycerides, AST
Serum muscle
enzymes
Serum
muscle
enzymes
ALT, GGT, Serum
muscle enzymes
AST, ALT, GGT
AST, ALT, GGT
GGT, transient
increase of serum
transaminases
Others
No
Umbilical hernia
Short stature,
peculiar facial
appearance
Mild lateral
facial
weakness
No
Short stature
No
Looking rather
older than his age
Reported by
N. Ronchetti
Z. EI-Kabbany
M.L. Williams
M.L. Williams
M.R. Judge
T. Kakourou
T. Kakourou
D. Mela
y, years; m, months; NE: not examined; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, g-glutamyl transpeptidase; CPK, creatine phosphokinase; SGOT, serum glutamic oxaloacetic transaminase;
SGPT, serum glutamic pyruvic transaminase; CNS, central nervous system; NCIE, nonbullous congenital ichthyosiform erytrhoderma Page 4 of 11 Page 4 of 11 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 spanning respectively 251 and 245 nt. The first promoter
region was amplified using 1F/1R primers, spanning from
-313 nt of the ABDH5 promoter to 338 nt of ABDH5
exon 1. A nested PCR was performed, using aF/aR and
bF/bR primer pairs, to analyze the second putative
promoter region. Patients and methods
Families and Specimens The PCR products were electrophoresed on a 2%
agarose gel containing ethidium bromide and their sizes
compared with those of the corresponding ABHD5
cDNA fragments from control subjects under a UV illu-
minator. RT-PCR products of CDS patients were
sequenced. ABHD5/CGI58 Mutations DNA sequence analysis of the putative promoter
regions, of the seven coding exons and of the exon-
intron boundaries of ABHD5 gene was performed in
eight CDS patients and their relatives. Five different
mutations were found in the six families from the Medi-
terranean area (Table 2). 5 CDS patients, A-II-1, B-II-1,
C-II-2, C-II-1 and D-II-2, were homozygotes for
ABHD5 mutations, and two patients, E-II-1 and E-II-2,
were compound heterozygotes. No ABHD5 mutations
were identified in patient, F-II-1. The new sequences
were submitted to GenBank (accession numbers are
shown in Table 2). All identified mutations segregated
within families. The wild-type ABHD5 genomic
sequence was extracted from GenBank accession num-
ber NG_007090.3. In the two families of Greek (E) and
Greek-Cypriot origin (D), two new genomic rearrange-
ments were detected. Patient D-II-2 showed the
c.898_*320del mutation resulting in premature termina-
tion of the protein, p.I300X (Additional file 3 Figure S2). Sequence analysis revealed a 1058 deletion that removed
63 bp of exon 6, intron 6 and exon 7 (Figure 2A). Although the D and E CDS families are not known to
be related, we found the same c.898_*320del mutation
in E-II-1 and E-II-2 patients, providing evidence for the
existence of a distal common ancestor. E-II-1 and E-II-2
subjects inherited this deletion maternally and another
new large deletion paternally, the c.662-1330_773+46del
mutation (Figure 2B). The last rearrangement occurred
within intron 4 and removed part of intron 4, exon 5
and part of intron 5. ABHD5 cDNA encompassing
exons 4, 5, 6 and 7 was examined by RT-PCR primers
in control, heterozygous parents (E-I-1, E-I-2) and
patients’ samples (E-II-1, E-II-2) (Figure 3A). Normal
(576 bp) and aberrant PCR products (464 and 277 bp)
were excised and sequenced, showing that one aberrant
band resulted from the exon 5 skipping and the second
one from skipping of both exons 5 and 6 (Figure 3B). The ABHD5 mRNA lacking exon 5 was the most com-
mon transcript in E-II-1 and E-II-2 patients. The aber-
rantly spliced mRNA would be expected to result in the
production of a protein lacking 129 amino acids in the
C-terminal region of ABHD5 (p.G221VfsX9). Another
RT-PCR product of about 400 bp was present in E-II-1,
E-II-2 and control subjects; it consisted of non-specific
sequence (Figure 3A). In the CDS patient from Molise, A-II-1, a novel homo- Figure 1 Lipid droplets images obtained from CDS patients. Results
Patients All the PCR
products were purified (NucleoSpin Extract II; M-Medi-
cal) and sequenced on 3730 DNA Analyzers (Applied
Biosystems, Foster City, CA) by the BigDye Terminator
V1.1 Cycle Sequencing Kit (Applied Biosystems). Reverse-Transcriptase PCR (RT-PCR) and cDNA analysis
Total RNA (1 μg) isolated from whole blood by the TRI-
zol method (Invitrogen, Carslbad, CA) was converted to
cDNA by RT-PCR using random hexamers (0.5 μg), 400
units of MMLV-RT, 1.6 mM total dNTPs, 20 units of
Rnasin, 0.4 mM dithiothreitol, in 50 μl of reaction solu-
tion containing 10 × RT Buffer. Five microliters of cDNA
was used to perform PCR amplification using 8F/8R and
9F/9R primer pairs designed to produce overlapping frag-
ments covering the entire sequence of the ABHD5 tran-
script (GenBank accession number AF151816). PCR
conditions for 8F/8R primers were as follows: denatura-
tion at 96°C for 3 min, annealing at 64°C for 40 sec and
extension at 72°C for 1 min for the first round, denatura-
tion at 95°C for 40 sec, annealing at 64°C for 40 sec and
extension at 72°C for 1 min for 35 cycles; denaturation at
95°C for 40 sec, annealing at 64°C for 40 sec and terminal
extension at 72°C for 3 min for the last cycle. For this
PCR reaction the DyNAzyme EXT was used. PCR condi-
tions for 9F/9R primer pairs consisted of an initial dena- Page 5 of 11 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
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http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 MGG and fluorescence detection of LDs in leukocytes and
fibroblasts from CDS patients MGG and fluorescence detection of LDs in leukocytes and
fibroblasts from CDS patients size of LDs in CDS fibroblasts were consistently higher
than in control cells. Large lipid vacuoles persisted
through multiple passages and were present even when
the CDS fibroblasts were grown in lipid-free media
(data not shown). In the peripheral buffy coat smears of all CDS patients,
stained with MGG and NR, we identified a variable per-
centage of Jordans’ bodies in neutrophilic and eosino-
philic granulocytes and in monocytes (Figure 1A). Examination of peripheral blood smears demonstrated
prominent cytoplasmic vacuoles in virtually every granu-
locyte and monocyte of C-II-2 and C-II-1 patients. Eighty to ninety percent of neutrophils, eosinophils and
basophils contained multiple LDs, as did monocytes
obtained from the remaining CDS patients (A-II-1, B-II-
1, D-II-2, E-II-1 and E-II-2), with the exception of F-II-
1, for whom lipid vacuoles were present in only 25% of
granulocytes and monocytes. Semi-confluent CDS and
control cultured fibroblasts were observed under phase-
contrast light microscopy and stained with NR prior to
fluorescence microscopy (Figure 1B). The number and ABHD5/CGI58 Mutations Direct sequencing of the 215 bp RT-PCR pro-
duct confirmed that A-II-1 cDNA contained the entire
intron 1 sequence (Additional file 5 Figure S4C). The
c.47+1G>A mutation resulted in a truncation of
the ABHD5 ORF at a premature stop codon located at
the beginning of intron 1. The mutated protein is pre-
dicted to consist of only 17 amino acids (pS17fsX1). RT-PCR product was obtained from A-II-1 cDNA
amplified with the 2F/2aR primers, showing that intron
1 was not eliminated during RNA splicing in this
patient. Direct sequencing of the 215 bp RT-PCR pro-
duct confirmed that A-II-1 cDNA contained the entire
intron 1 sequence (Additional file 5 Figure S4C). The
c.47+1G>A mutation resulted in a truncation of
the ABHD5 ORF at a premature stop codon located at
the beginning of intron 1. The mutated protein is pre-
dicted to consist of only 17 amino acids (pS17fsX1). a splice-site mutation affecting the invariant G of the
intron-1 donor splice-site GT dinucleotide (Additional
file 4 Figure S3A). This splice-site mutation is expected
to lead to aberrant splicing with retention of intron 1;
this was confirmed by RT-PCR (Additional file 5 Figure
S4A). Using the 2F/2aR primer pairs, no amplification
product was expected from control cDNA since the for-
ward primer (2F) localizes within intron 1. A 215 bp a splice-site mutation affecting the invariant G of the
intron-1 donor splice-site GT dinucleotide (Additional
file 4 Figure S3A). This splice-site mutation is expected
to lead to aberrant splicing with retention of intron 1;
this was confirmed by RT-PCR (Additional file 5 Figure
S4A). Using the 2F/2aR primer pairs, no amplification
product was expected from control cDNA since the for-
ward primer (2F) localizes within intron 1. A 215 bp Figure 2 Novel ABHD5 genomic rearrangements identified in
CDS families. A Sequence analysis showing c.898_*320del
mutation. B Sequence analysis showing the c.662-1330_773+46del
mutation. Figure 3 Molecular characterization of the c.662-1330_773
+46del in CDS family E. A, RT-PCR performed with primers
encompassing exons 4, 5, 6 and 7, showing absence of wild-type
product (546 bp) in E-II-1 and E-II-2 patients and the presence of a
dominant RT product of 464 bp and a minor product of 277 bp
resulting from the skipping of exon 5 and of exons 5 and 6,
respectively. Lane M: 100-bp molecular weight marker. Lanes II-1
and II-2: CDS patients. ABHD5/CGI58 Mutations A
Buffy coats from CDS patients; A1,2 Microphotographs of May-
Grünwald-Giemsa and A3,4 of Nile red (NR) and DAPI-stained buffy
coats. Scale bar: 10 μm. B Cultured fibroblasts from control (B1, B3)
and affected (B2, B4) patients. Phase contrast images: B1,2. Fluorescent microscopy images with Nile red staining: B3,4. Scale
bar: 40 μm. Figure 1 Lipid droplets images obtained from CDS patients. A
Buffy coats from CDS patients; A1,2 Microphotographs of May-
Grünwald-Giemsa and A3,4 of Nile red (NR) and DAPI-stained buffy
coats. Scale bar: 10 μm. B Cultured fibroblasts from control (B1, B3)
and affected (B2, B4) patients. Phase contrast images: B1,2. Fluorescent microscopy images with Nile red staining: B3,4. Scale
bar: 40 μm. In the CDS patient from Molise, A-II-1, a novel homo-
zygous mutation, the c.47+1G>A, was detected. This is Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Page 6 of 11 Table 2 ABHD5 gene mutations
Patients
DNA position
cDNA o DNA mutation
Protein mutation
GenBank accession numbera
A-II-1
IVS 1
c.47+1G>A
p.S17fsX1
HM474790
B-II-1
E 5
c.700C>T
p.R234X
/
C-II-2
IVS 6
c.960+5G>A
p.A321VfsX10
HM474791
C-II-1
IVS 6
c.960+5G>A
p.A321VfsX10
HM474791
D-II-2
E 6/IVS 6/E 7
c.898_*320del
p.I300X
HM474793
E-II-I
E 6/IVS 6/E 7
IVS 4/E 5/IVS 5
c.[898_*320del]+
[662-1330_773+46del]
p.[I300X]+
[G221VfsX9]
HM474793;
HM474792
E-II-2
E 6/IVS 6/E 7
IVS 4/E 5/IVS 5
c.[898_*320del]+
[662-1330_773+46del]
p.[I300X]+
[G221VfsX9]
HM474793;
HM474792
cDNA numbering begins with +1 as the A of the translation initiation codon; the translation initiator methionine is numbered as +1; novel mutations are typed
in bold; E, exon; IVS, intervening sequence; c, cDNA; g, gene. a GenBank accession number of the new mutations identified in this study Table 2 ABHD5 gene mutations cDNA numbering begins with +1 as the A of the translation initiation codon; the translation initiator methionine is numbered as +1
in bold; E, exon; IVS, intervening sequence; c, cDNA; g, gene. cDNA numbering begins with +1 as the A of the translation initiation codon; the translation initiator methionine is numbered as +1; novel mutations are typed
in bold; E, exon; IVS, intervening sequence; c, cDNA; g, gene. a GenBank accession number of the new mutations identified in this study RT-PCR product was obtained from A-II-1 cDNA
amplified with the 2F/2aR primers, showing that intron
1 was not eliminated during RNA splicing in this
patient. ABHD5/CGI58 Mutations While the prox-
imal
breakpoint
of
c.898_*320del
mutation
(g.31913_32970del1058) is not located within any
repeat sequence, its distal breakpoint lays within a X7B
LINE retrotransposon which belongs to the long inter-
spersed elements LINE-1 (or L1) (g.32956_33063). Moreover, the presence of a micro-homology of 3 bp
plus 3 bp (i.e. TGC and TAG) in the junction
sequence/breakpoints accounts for the model of repli-
cation slippage [26] (Figure 4A). The second large
deletion
c.662-1330_773+46del
(g.277
35_29222
del1487) is also associated in cis to a 18 bp (g.27
698_27699ins18) insertion and a nucleotide deletion
(g.27730delG), which are not reported as ABHD5 poly-
morphisms (Figure 4B). The c.662-1330_773+46del
proximal breakpoint is near an Alu sequence, while its
distal breakpoint is located within a TG-simple repeat
and very close to an Alu element (distance: 75 bp). Inside the deleted region there is another Alu
sequence which is lost in the mutated allele. Alu
repeat density in this region of ABHD5 gene (27010-
29611) is very high. These Alu elements belong to the
Alu-Sx subfamily and, like other repetitive sequences,
have been shown to be involved in molecular
mechanisms leading to rearrangements in the human
genome [27]. The data show that the c.662-1330_773
+46del mutation should be considered as a complex
gene rearrangement. This is probably due to a differ-
ent mutational mechanism in comparison with that
which occurred for the c.898_*320del mutation. The
consequences of gene deletions have been investigated
through an exhaustive analysis of normal and aber-
rant ABHD5 mRNAs. These deletions may lead to
shorter ABHD5 proteins, pG221VfsX9 and pI300X,
lacking 139 and 50 COOH-terminal amino acids,
respectively. A novel mutation was also detected in the C-II-2 and
C-II-1 patients from Palestine. This homozygous splice-
donor-site mutation, c.960+5G>A, caused abnormal
RNA splicing (Additional file 4 Figure S3B). The region
of the ABHD5 transcript, including exons 6 and 7, was
amplified by RT-PCR. Instead of the expected cDNA
fragment (236 bp), a longer cDNA fragment of 821 bp
in size was detected (Additional file 5 Figure S4B). Sequence analysis showed that the longer cDNA pro-
duct arose from abnormal retention of intron 6 (Addi-
tional file 5 Figure S4D). The c.960+5G>A splice-site
mutation creates a premature stop codon within intron
6 and removes 29 amino acids from the C-terminal tail
of the protein (p.A321VfsX10). p
p
In the Egyptian family, the p.R234X mutation was
present. ABHD5/CGI58 Mutations Lane I-1: father, carrying the c.662-1330_773
+46del mutation in heterozygous state. Lane I-2: mother, carrying
the other deletion. Lane C: control. B, Electropherograms of 464 bp
and 277 bp abnormal RT-PCR products. Figure 2 Novel ABHD5 genomic rearrangements identified in
CDS families. A Sequence analysis showing c.898_*320del
mutation. B Sequence analysis showing the c.662-1330_773+46del
mutation. Figure 3 Molecular characterization of the c.662-1330_773
46d l i
CDS f
il
E A RT PCR
f
d
ith
i Figure 3 Molecular characterization of the c.662-1330_773
+46del in CDS family E. A, RT-PCR performed with primers Figure 3 Molecular characterization of the c.662-1330_773
+46del in CDS family E. A, RT-PCR performed with primers
encompassing exons 4, 5, 6 and 7, showing absence of wild-type
product (546 bp) in E-II-1 and E-II-2 patients and the presence of a
dominant RT product of 464 bp and a minor product of 277 bp
resulting from the skipping of exon 5 and of exons 5 and 6,
respectively. Lane M: 100-bp molecular weight marker. Lanes II-1
and II-2: CDS patients. Lane I-1: father, carrying the c.662-1330_773
+46del mutation in heterozygous state. Lane I-2: mother, carrying
the other deletion. Lane C: control. B, Electropherograms of 464 bp
and 277 bp abnormal RT-PCR products. +46del in CDS family E. A, RT PCR performed with primers
encompassing exons 4, 5, 6 and 7, showing absence of wild-type
product (546 bp) in E-II-1 and E-II-2 patients and the presence of a
dominant RT product of 464 bp and a minor product of 277 bp
resulting from the skipping of exon 5 and of exons 5 and 6,
respectively. Lane M: 100-bp molecular weight marker. Lanes II-1
and II-2: CDS patients. Lane I-1: father, carrying the c.662-1330_773
+46del mutation in heterozygous state. Lane I-2: mother, carrying
the other deletion. Lane C: control. B, Electropherograms of 464 bp
and 277 bp abnormal RT-PCR products. Figure 2 Novel ABHD5 genomic rearrangements identified in
CDS families. A Sequence analysis showing c.898_*320del
mutation. B Sequence analysis showing the c.662-1330_773+46del
mutation. Page 7 of 11 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 (Institute for System Biology, Seattle, WA, USA) was
used and the repeat elements or sequence homologies
around the breakpoints were analyzed. ABHD5/CGI58 Mutations The same mutation had been identified in an
adult case of CDS by Schleinitz et al. [25]. None of the
novel mutations, identified in CDS patients, were
observed in more than 100 alleles from control sub-
jects. Finally, RT-PCR analysis and sequencing of full-
length ABHD5 cDNA was performed in F-II-1 patient
(Sicily) for which no genomic mutations were found. This result confirmed the negative DNA analysis and
excluded distant intronic mutations that might have
affected mRNA splicing. Furthermore, direct sequen-
cing of two putative promoter regions upstream the
ATG starting codon of ABHD5 gene failed to detect
any pathogenic mutation. Discussion
h
d Since the identification of the ABHD5/CGI58 gene and
the detection of its mutations in nine CDS families by
Lefèvre et al [13], CDS (NLSD with ichthyosis) has been
considered to be a unique clinical variant of NLSD with
a defined genetic cause. ABHD5 is a co-activator of
ATGL, a novel lipase that catalyses the initial step of
TG hydrolysis in adipocyte and non-adipocyte LDs [15]. These data suggest an important biochemical role for
ABHD5 in the intracellular catabolism of neutral lipids
and provide an explanation for the pathogenic effects of
ABHD5 mutations. In addition, ABHD5 is a lysopho-
sphatidic acid acyltransferase; the relationship of this
activity to the clinical phenotype remains unclear
[16,17]. The splicing errors identified in our CDS families all
represent novel mutations. They are homozygous G to
A transitions which occurred in the splice consensus
motifs of introns 1 and 6. In patient A-II-1 from Molise,
the c.47+1G>A mutation eliminates approximately 96%
of the ABHD5 protein. The consequence of this muta-
tion may lead to the complete absence of the mutated
protein, through protein instability. The E-II-2 and E-II-
3 patients of Palestinian origin presented with the c.960
+5G>A mutation. This mutation is expected to give rise
to a truncated protein lacking 28 amino acids at the C-
terminal region of ABHD5 (consisting of 320 out of 349
amino acids). Our study extends the spectrum of ABHD5 disease-
causing mutations in CDS. Sequence analysis reveals
five different mutations distributed all along the
ABHD5 gene in eight CDS patients from six families
from the Mediterranean area. The identified genetic
variations include one nonsense mutation, two splice-
site substitutions and, for the first time, two large dele-
tions. In order to verify whether these large genomic
rearrangements could be explained by a common
mutational mechanism, the RepeatMasker Software In the Egyptian patient (B-II-1), the R234X homozy-
gous mutation conserves 67% of the ABHD5 wild-type
protein. This patient was a one-year-old boy with Page 8 of 11 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Figure 4 Diagram of the two large deletions identified in CDS-D and CDS-E families. A, Diagram of the 1058 bp deletion found in D-II-1,
E-II-2 and E-II-1 patients. Normal sequences at the 5’ and 3’ breakpoints of the deletion are aligned with the deleted sequence. The 6 bp micro-
homology at the breakpoints is highlighted in grey. Discussion
h
d Part of the Alu sequence at the 3’-breakpoint is underlined. The structure of the abnormal
allele in the region of the deletion is shown at the bottom of the diagram. B, Diagram of the 1487 bp deletion identified in E-II-2 and E-II-1
patients. The breakpoints, reported on the model, are inside intron 4 and inside a GT repeat (black area) in intron 5. The site of the 18 bp
insertion is also reported. It is in very close proximity of the breakpoint. Figure 4 Diagram of the two large deletions identified in CDS-D and CDS-E families. A, Diagram of the 1058 bp deletion found in D-II-1,
E-II-2 and E-II-1 patients. Normal sequences at the 5’ and 3’ breakpoints of the deletion are aligned with the deleted sequence. The 6 bp micro-
homology at the breakpoints is highlighted in grey. Part of the Alu sequence at the 3’-breakpoint is underlined. The structure of the abnormal
allele in the region of the deletion is shown at the bottom of the diagram. B, Diagram of the 1487 bp deletion identified in E-II-2 and E-II-1
patients. The breakpoints, reported on the model, are inside intron 4 and inside a GT repeat (black area) in intron 5. The site of the 18 bp
insertion is also reported. It is in very close proximity of the breakpoint. generalized ichthyosis, bilateral ectropion, hepatospleno-
megaly (noted at the age of 9 months), myopathy and
an umbilical hernia. The same mutation had been pre-
viously identified in a 42-year-old man from France,
who was heterozygous for R284X and H82R [25]. Despite delayed confirmation of the diagnosis, this
patient presented with typical symptoms of CDS, includ-
ing NCIE, muscle weakness, bilateral sub-capsular cataracts and neurosensory hearing loss. He did not
have liver dysfunction or CNS abnormalities. Some
important clinical differences emerged between the
Egyptian and French patients, concerning, in particular,
the hepatic involvement. These differences might be due
to homozygous versus heterozygous conditions or by
modifier genes and epigenetic factors which might be
involved in these variations. Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Page 9 of 11 All the mutations described in this work are predicted
to result in truncated proteins that lack different
ABHD5 regions (Additional file 6 Figure S5). Discussion
h
d In spite of
our efforts, it remains very difficult to find a correlation
between the phenotypic and genotypic characteristics,
since most of ABHD5 mutations are novel and unique. Surprisingly, we noticed that the c.960+5G®A muta-
tion, which conserves 92% of the native protein, was
associated with patients severely affected by neurological
symptoms, hearing loss and myopathy (C-II-2, C-II3),
whereas the c.47+1G>A mutation that caused a dra-
matic truncation of the ABHD5 protein (p.S17X), was
associated with severe steatohepatitis but a relatively
mild phenotype concerning the other clinical CDS fea-
tures (patient: A-II-1). It is possible that the accumula-
tion of non-functional ABHD5 proteins has a more
deleterious consequence for cellular LD metabolism in
some tissues than total loss of the protein expression. However, any explanation of this phenomenon remains
highly speculative, since the precise roles of the ABHD5
domains remain largely unknown. Nevertheless, we can
postulate that the consequence of the c.47+1G>A muta-
tion (p.S17X) would be similar to classical loss of func-
tion or a knockout mutation, as the residual peptide
contains only 17 of the 349 amino acids of the wild-type
protein. We were unable to identify any mutation in ABHD5
in patient F-II-1. When this patient was first examined
at the age of 16, his health was good, although he had
congenital ichthyosis, hepatomegaly, a persistent
increase of GGT and a transient increase of transami-
nase, with no history of drug or alcohol abuse. On the
peripheral blood smear, LDs were detected in only
20-30% of neutrophilic and eosinophilic granulocytes
and in monocytes. On the basis of the clinical and histo-
logical findings, F-II-1 was diagnosed as having CDS
[19]. To provide an exhaustive analysis of the ABHD5
gene in this patient, we screened the putative promoter/
regulatory regions but failed to find any variation. The
absence of any mutation in the ABHD5 gene in F-II-1
suggests that the etiology of CDS is genetically heteroge-
neous. Similar to our results, in one family of Algerian
origin presenting with a CDS phenotype, Lefèvre et al. [13] identified two regions of homozygosity, one consist-
ing of 11 cM on chromosome 3p21 (containing the
ABHD5 gene), and the other spanning 18 cM on chro-
mosome 14; this report and our evidence point to possi-
ble genetic heterogeneity of the syndrome. Although the
F-II-1 patient had no evidence of myopathy, we also
sequenced the ATGL gene but found no mutation. Discussion
h
d CDS arises from a defect of LD metabolism leading to
a systemic increase in the size and number of these
cytosolic inclusions. Despite their classic denomination
as simple lipid structures, LDs are complex and highly
dynamic organelles [31] with a large complement of
associated proteins, including scaffold proteins, lipases
and co-activators, which have been found to change
their associations with lipid droplets in response to lipo-
lytic stimulation. Relatively little is known about tem-
poral and spatial relationships among these LD proteins. Further studies are needed to identify new candidate
genes involved in NLSDs, and further extensive ABHD5
gene analysis in a larger panel of CDS families would be
useful in order to gain additional insights into the varia-
bility of clinical expression and the factors contributing
to CDS. p
The N-terminal region ABHD5 (1-30 amino acids) is
essential for correct localization to the lipid droplet and
ABHD5 lacking this amino acid region, loses the ability
to activate ATGL [28]. Four of the protein variants
retain the hydrophobic motif (Additional file 6: Figure
S5) but lack the HX4D motif, between amino acids 327
and 332, specific for proteins with acyltransferase activ-
ity [16]. Moreover, only 2 mutants retain both Q130
and E260, amino acid residues were previously identified
as essential for ABHD5-perilipin interaction as well as
for ATGL activation [15,29]. ABHD5 is a binding part-
ner for perilipin and ADRP, two proteins of the PAT-
domain family associated with the surface of LDs. Using
double-label immunocytochemistry, Granneman et al. [30] found that perilipin acts as a link for ABHD5 when
adipocytes are in the basal state. After protein kinase A
(PKA) phosphorylation, perilipin decreases its interaction
with ABHD5 and ABHD5 increases its co-localization
with ATGL. One of the most important issues
that remains to be solved is to identify ABHD5 regions
(epitopes) that specifically interact with perilipin or with
other proteins. Structure and function analysis of
mutated ABHD5 proteins should be performed in order
to investigate potential intra-molecular interactions
between epitopes, as well as binding affinity to LDs
proteins. References 1. Rozenszajn L, Klajman A, Yaffe D, Efrati P: Jordans’ anomaly in white blood
cells. Report of case. Blood 1996, 28:258-265. 1. Rozenszajn L, Klajman A, Yaffe D, Efrati P: Jordans’ anomaly in white blood
cells. Report of case. Blood 1996, 28:258-265. Additonal file 5: Supplementary Figure 4. Molecular characterization
of the c.47+1G>A and c.960+5G>A ABHD5 mutations. A, RT-PCR of part
of intron 1 and exon 2 from cDNA of the control subject (no
amplification product) and A-II-1 patient (215 bp); Lane 1: 100-bp
molecular weight marker. B, RT-PCR of exons 6 and 7 from cDNA of a
control subject (236 bp) and C-II-2 or C-II-3 patient (821 bp); Lane 1: 100-
bp molecular weight marker. C, Partial sequences of exon1/exon2 from
cDNA of a control subject and of intron1/exon2 from cDNA of the A-II-1
patient. D, Partial sequences of exon6/7 from cDNA of a control subject
and of exon6/intron6 from cDNA of the C-II-2 or C-II-3 patients. Additonal file 5: Supplementary Figure 4. Molecular characterization
of the c.47+1G>A and c.960+5G>A ABHD5 mutations. A, RT-PCR of part
of intron 1 and exon 2 from cDNA of the control subject (no
amplification product) and A-II-1 patient (215 bp); Lane 1: 100-bp
molecular weight marker. B, RT-PCR of exons 6 and 7 from cDNA of a
control subject (236 bp) and C-II-2 or C-II-3 patient (821 bp); Lane 1: 100-
bp molecular weight marker. C, Partial sequences of exon1/exon2 from
cDNA of a control subject and of intron1/exon2 from cDNA of the A-II-1
patient. D, Partial sequences of exon6/7 from cDNA of a control subject
and of exon6/intron6 from cDNA of the C-II-2 or C-II-3 patients. 2. Dorfman ML, Hershko C, Eisenberg S, Sagher F: Ichthyosiform dermatosis
with systemic lipidosis. Arch Dermatol 1974, 110:261-266. with systemic lipidosis. Arch Dermatol 1974, 110:261-266. 3. Chanarin I, Patel A, Slavin G, Wills EJ, Andrews TM, Stewart G: Neutral-lipid
storage disease: a new disorder of lipid metabolism. Br Med J 1975,
1:553-555. 4. Takahira T, Utsunomiya T, Ishijima M, Mori H, Yano K, Nunobiki T, Eto H:
Specific myocardial disease caused by multisystemic triglyceride storage
in Jordans’ anomaly. Am Heart J 1993, 126:995-997. y
,
5. Igal RA, Rhoads JM, Coleman RA: Neutral lipid storage disease with fatty
liver and cholestasis. J Pediatr Gastroenterol Nutr 1997, 25:541-547. Additional file 6: Supplementary Figure 5. Domain organization of
wild-type and mutant ABHD5 variants. Conclusions Our ABHD5 mutational analysis extends the molecular
genetic heterogeneity of CDS. We have identified new
splice site mutations and, for the first time, novel large
deletions, demonstrating that sequencing, long range
PCR and RT-PCR analysis are necessary to perform a
complete molecular screening for ABHD5 gene muta-
tions in order to avoid allelic drop-out phenomena. Moreover, our findings contribute to understanding of
the complex effects that different ABHD5 gene muta-
tions may have on the CDS phenotype. Page 10 of 11 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Biochemistry and Clinical Biochemistry, Catholic University, Gemelli Hospital,
Rome, Italy. Acknowledgements The authors are grateful to all patients and their families. We would like to
thank GianPaolo Martelli for graph support and Danny D’Agostini, Gloria
Invernici, Silvia Cristini, Dario DeGiorgio, Elisa Colombo and Sara Missaglia for
generous scientific and technical assistance. This study was financed by
grants from the Cariplo Foundation (Milan, Italy), from COPEV (Comitato per
la Prevenzione dell’Epatite Virale, Milan, Italy) and from the US National
Institutes of Health (DK56598). The authors are grateful to all patients and their families. We would like to
thank GianPaolo Martelli for graph support and Danny D’Agostini, Gloria
Invernici, Silvia Cristini, Dario DeGiorgio, Elisa Colombo and Sara Missaglia for
generous scientific and technical assistance. This study was financed by The authors are grateful to all patients and their families. We would like to
thank GianPaolo Martelli for graph support and Danny D’Agostini, Gloria
Invernici, Silvia Cristini, Dario DeGiorgio, Elisa Colombo and Sara Missaglia for
generous scientific and technical assistance. This study was financed by
grants from the Cariplo Foundation (Milan, Italy), from COPEV (Comitato per
la Prevenzione dell’Epatite Virale, Milan, Italy) and from the US National
Institutes of Health (DK56598). 13. Lefevre C, Jobard F, Caux F, Bouadjar B, Karaduman A, Heilig R, Lakhdar H,
Wollenberg A, Verret JL, Weissenbach J, Ozguc M, Lathrop M,
Prud’homme JF, Fischer J: Mutations in CGI-58, the gene encoding a new
protein of the esterase/lipase/thioesterase subfamily, in Chanarin-
Dorfman syndrome. Am J Hum Genet 2001, 69:1002-1012. grants from the Cariplo Foundation (Milan, Italy), from COPEV (Comitato per
la Prevenzione dell’Epatite Virale, Milan, Italy) and from the US National
Institutes of Health (DK56598). 14. Bruno C, Bertini E, Di Rocco M, Cassandrini D, Ruffa G, De Toni T, Seri M,
Spada M, Di Volti G, D’Amico A, Trucco F, Arca M, Casali C, Angelini C,
DiMauro S, Minetti C: Clinical and genetic characterization of Chanarin-
Dorfman syndrome. Biochem Biophys Res Com 2008, 369:1125-1128. Abbreviations CDS: Chanarin-Dorfman syndrome; CNS: central nervous system; FA: fatty
acid; GGT: g-glutamyl transpeptidase; LD: lipid droplet; MGG: May-Grünwald-
Giemsa; NCIE: nonbullous congenital ichthyosiform erythroderma; NLSDs:
Neutral-lipid storage diseases; NR: Nile Red; PKA: protein kinase A; TG:
triacylglycerol. 11. Igal RA, Coleman RA: Acylglycerol recycling from triacylglycerol to
phospholipid, not lipase activity, is defective in neutral lipid storage
disease fibroblasts. J Biol Chem 1996, 271:16644-16651. 12. Igal RA, Coleman RA: Neutral lipid storage disease: a genetic disorder
with abnormalities in the regulation of phospholipid metabolism. J Lipid
Res 1998, 39:31-43. Competing interests
Th
h
d
l
h The authors declare that they have no competing interests. Additional file 4: Supplementary Figure 3. ABHD5 novel splice-site
mutations identified in the A and C CDS families. A, mutation affecting
the invariant G of the donor splice-site of intron 1 (c.47+1G>A) in A-II-1. B, mutation in the conserved donor splice-site of intron 6 (c.960+5G>A)
in C-II-2 and C-II-3. Arrowheads indicate the positions of the mutations in
affected patients. Lane 1: 100-bp molecular weight marker. Received: 26 August 2010 Accepted: 1 December 2010
Published: 1 December 2010 Received: 26 August 2010 Accepted: 1 December 2010
Published: 1 December 2010 Received: 26 August 2010 Accepted: 1 December 2010
Published: 1 December 2010 References The ABHD5 theoretical variants
resulting from the six mutations identified in this study are truncated
proteins lacking different portions of wild-type ABHD5. Five of the six
mutant variants retained the hydrophobic motif, located between
residues 69 and 87 (dark-grey area), that represents the putative lipid-
binding domain. However, all six mutant proteins lacked the HX4D motif
between amino acids 327 and 332, specific for proteins with
acyltransferase activity. Q130 and E260, reported in the models, have
previously been identified as essential residues for ABHD5-perilipin
interaction and for ATGL activation. 6. Wollenberg A, Schaller M, Roschinger W, Schirren CG, Wolff H: Dorfman-
Chanarin syndrome - eine neutrallipidspeicher-krankheit. Hautarzt 1997,
48:753-758. 6. Wollenberg A, Schaller M, Roschinger W, Schirren CG, Wolff H: Dorfman-
Chanarin syndrome - eine neutrallipidspeicher-krankheit. Hautarzt 1997,
48:753-758. 7. Ronchetti N, Prati D, Pezzotta MG, Tavian D, Colombo R, Callea F, Colli A:
Severe steatohepatitis in a patient with a rare neutral lipid storage
disorder due to ABHD5 mutation. J Hepatol 2008, 49:474-477. 8. Ciesek S, Hadem J, Fischer J, Manns MP, Strassburg CP: A rare cause of
nonalcoholic fatty liver disease. Ann Intern Med 2006, 145:154-155. acyltransferase activity. Q130 and E260, reported in the models, have
previously been identified as essential residues for ABHD5-perilipin
interaction and for ATGL activation. 9. Williams ML, Coleman RA, Placezk D, Grunfeld C: Neutral lipid storage
disease: a possible functional defect in phospholipid-linked
trialcilglycerol metabolism. Biochim Biophys Acta 1991, 1096:162-169. 10. Hilaire N, Salvayre R, Thiers JC, Bonnafe MJ, Negre-Salvayre A: The turnover
of cytoplasmic triacylglycerols in human fibroblasts involves two
separate acyl chain length-dependent degradation pathways. J Biol
Chem 1995, 270:27027-27034. Authors’ contributions Additional file 1: Supplementary Figure 1. Pedigrees of the CDS
families. CR carried out the molecular genetic studies and the interpretation of the
results. RAC and LM made substantial contributions to interpretation of data
and participated in manuscript preparation. CDV, SME, DP, AC and RC were
involved in the clinical evaluation of patients and manuscript revision. DT
made substantial contributions to conception, analysis and interpretation of
data and drafted the manuscript. All authors read and approved the final
manuscript. Additional file 2: Supplementary Table 1. Primers for genomic and
cDNA analysis of ABHD5 gene. Additional file 2: Supplementary Table 1. Primers for genomic and
cDNA analysis of ABHD5 gene. Additional file 3: Supplementary Figure 2. PCR products obtained
utilizing 6F/7R primers in a control and the D-II-2 patient. While an
expected band of 1338 bp was present in the control sample, a 280 bp
product was detected in the CDS patient. The shorter PCR product
differs from control for about 1050 bp. Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 is a coenzyme A-dependent lysophosphatidic acid acyltransferase. J Lipid
Res 2010, 51:709-719. is a coenzyme A-dependent lysophosphatidic acid acyltransferase. J Lipid
Res 2010, 51:709-719. 18. Fischer J, Lefevre C, Morava E, Mussini JM, Laforet P, Negre-Salvayre A,
Lathrop M, Salvayre R: The gene encoding adipose triglyceride lipase
(PNPLA2) is mutated in neutral lipid storage disease with myopathy. Nat
Genet 2007, 39:28-30. 19. Mela D, Artom A, Goretti R, Varagona G, Riolfo M, Ardoino S, Sanguineti G,
Vitali A, Ricciardi S: Dorfman-Chanarin syndrome: a case with prevalent
hepatic involvement. J Hepatol 1996, 25:769-771. 20. El-Kabbany Z, Elsayed SM, Rashad M, Tareef R, Galal N: Dorfman-Chanarin
syndrome in Egypt. Am J Med Genet 2003, 121A:75-78. syndrome in Egypt. Am J Med Genet 2003, 121A:75-78 21. Williams ML, Koch TK, O’Donnell JJ, Frost PH, Epstein LB, Grizzard WS, 21. Williams ML, Koch TK, O’Donnell JJ, Frost PH, Epstein LB, Grizzard WS,
Epstein CJ: Ichthyosis and neutral lipid storage disease. Am J Med Gen
1985, 20:711-726. Epstein CJ: Ichthyosis and neutral lipid storage disease. Am J Med Gen
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Winchester B, Lake BD: Neutral lipid storage disease. Case report and
lipid studies. Br J Dermat 1994, 130:507-510. 23. Kakourou T, Drogari E, Christomanou H, Giannoulia A, Dacou-Voutetakis C:
Neutral lipid storage disease-response to dietary intervention. Arch Dis
Child 1997, 77:184. 24. Tavian D, Colombo R: Improved cytochemical method for detecting
Jordans’ bodies in neutral-lipid storage diseases. J Clin Pathol 2007,
60:956-958. 25. Schleinitz N, Fischer J, Sanchez A, Veit V, Harle JR, Pelissier JF: Two new
mutations of the ABHD5 gene in a new adult case of chanarin dorfman
syndrome: an uncommon lipid storage disease. Arch Dermatol 2005,
141:798-800. 26. Chen JM, Chuzhanova N, Stenson PD, Férec C, Cooper DN: Meta-analysis
of gross insertion causing human genetic disease: novel mutational
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25:207-221. 27. Rossetti LC, Goodeve A, Larippa IB, De Brasi C: Homologous recombination
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2006, 3:309-319. 1Department of Psychology, Catholic University of the Sacred Heart, Milan,
Italy. 2Department of Nutrition, University of North Carolina, Chapel Hill, NC,
USA. 3DiSCAFF Department, University of Piemonte Orientale, Novara. 4Department of Paediatrics, Athens University, Greece. 5Medical Genetics
Center, Korba, Cairo, Egypt. 6Department of Transfusion Medicine and
Hematology, Ospedale Alessandro Manzoni, Lecco, Italy. 7Center of
Transfusion Medicine, Cellular Therapy and CryoBiology, IRCCS Foundation
Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. 8Department of
Internal Medicine, Ospedale Alessandro Manzoni, Lecco, Italy. 9Department
of Internal Medicine, Santa Corona Hospital, Pietra Ligure, Italy. 10Institute of 16. Ghosh AK, Ramakrishnan G, Chandramohan C, Rajasekharan : CGI-58, the
causative gene for Chanarin-Dorfman syndrome, mediates acylation of
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Xu Z, Lara-Gonzalez S, Storch J, Carman GM, Brasaemle DL: CGI-58/ABHD5 Page 11 of 11 Page 11 of 11 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
http://www.ojrd.com/content/5/1/33 Redaelli et al. Orphanet Journal of Rare Diseases 2010, 5:33
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CGI-58 mislocalization in Chanarin-Dorfman syndrome. J Biol Chem 2004,
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ovary K2 cell lipid droplets appear to be metabolic organelles involved
in membrane traffic. J Biol Chem 2004, 279:3787-3792. doi:10.1186/1750-1172-5-33
Cite this article as: Redaelli et al.: Clinical and genetic characterization of
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A Lacanian Approach to Medical Demand, With a Focus on Pediatric Genetics: A Plea for Subjectivization
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Frontiers in psychology
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Keywords: genetics, pediatrics, demand, subjectivization, medical knowledge, transference, desire, doctor-
patient relationship A Lacanian Approach to Medical
Demand, With a Focus on Pediatric
Genetics: A Plea for Subjectivization Rémy Potier1† and Olivier Putois2,3*† 1 Centre de Recherches Psychanalyse, Médecine et Société CRPMS (EA 3522), Université Paris Diderot, Paris, France,
2 Université de Strasbourg, SuLiSoM EA 3071, Strasbourg, France, 3 Department of Psychiatry, Mental Health and
Addictology, Strasbourg University Hospital, Strasbourg, France Current psychological research on contemporary medicine, and in particular genetics,
often targets the underpinnings of patients’ attitudes and behaviors with respect to
biomedical knowledge and healthcare practices. But few studies approach these
underpinnings as manifestations of the unconscious, while so doing could (in
particular) help understand patients’ apparent difficulties to understand information,
and to subsequently act accordingly (e.g., in making therapeutic decisions, etc.). We
hypothesize that Lacan’s (1966) remarks (“The place of psychoanalysis in medicine”) on
the transferential nature of the demand addressed by the patient (or his family) to the
doctor can help account for these issues: demand filters medical information received
from the practitioner, and thereby motivates subsequent decisions. In this paper, we try
and shed light on this thesis, and focus on pediatric genetics. We start by describing
the manifest doctor-patient-family relationship in the pediatric genetics consultation, in
order to show where unconscious determinants can come to play a role (1). We then
explain Lacan’s theory of demand: what the patient unknowingly demands is knowledge
(savoir), the object of which is the body of jouissance – the libidinal experience of
one’s body through the first libidinal exchanges with the Other of early infancy, whereby
the subject is assigned by the Other (subjectification) a specific fantasmatic status
organizing his desire. Patients’ understanding and attitudes thus vary so greatly because
of this pre-existing filter. Healing and cure are merely apparent objects of the medical
demand, which is an invocative drive seeking knowledge on the cause of one’s desire:
medical demand is an instance of transference. Doctors should thus enable patient
subjectivization, i.e., help them realize that their demand’s genuine object lies in their pre-
existing subjective coordinates (2). In pediatric genetics, apparently paradoxical family
attitudes heavily draw on what G. Raimbault, drawing on Lacan, called implicit demand,
the object of which is knowledge about the family fantasy giving shape to the guilt
of possibly transmitting the disease. We give a clinical example, then show how the
concept of demand helped us elaborate the core of a research project on the subjective
effects of a genetic deafblindness handicap (3). HYPOTHESIS AND THEORY
published: 01 November 2018
doi: 10.3389/fpsyg.2018.02021 Edited by:
Fabian Guénolé,
University of Caen Normandy, France Edited by:
Fabian Guénolé,
University of Caen Normandy, France Edited by:
Fabian Guénolé,
University of Caen Normandy, France Reviewed by:
Marjorie Roques,
Normandie Université, France
François Medjkane,
Centre Hospitalier Regional et
Universitaire de Lille, France
Kazushige Shingu,
Nara University, Japan
Pierre-Henri Castel,
Centre National de la Recherche
Scientifique (CNRS), France *Correspondence:
Olivier Putois
olivier.putois@gmail.com
†These authors have contributed
equally to this work as co-first authors Specialty section:
This article was submitted to
Psychoanalysis
and Neuropsychoanalysis,
a section of the journal
Frontiers in Psychology
Received: 03 October 2017
Accepted: 01 October 2018
Published: 01 November 2018
Citation:
Potier R and Putois O (2018) A
Lacanian Approach to Medical
Demand, With a Focus on Pediatric
Genetics: A Plea for Subjectivization. Front. Psychol. 9:2021. doi: 10.3389/fpsyg.2018.02021 INTRODUCTION taking therapeutic decisions, from short-term life-or-death
transplant to long-term therapeutic compliance. One of the main axes of contemporary psychological research
on healthcare and biomedicine revolves around the impact
of personalized medicine. This is especially true with respect
to medical genetics and genomics, which are undergoing an
exponential development. This development gives rise to new
problems, such as the use of unsolicited or secondary findings,
supplemental information unrelated to the patient’s initial
request, and yet of potentially crucial medical importance (such
as BRCA 1 or 2 – see, e.g., Christenhusz et al., 2013). While biomedical and genetic knowledge have developed
exponentially since Lacan’s (1966) lecture at the Salpêtrière
Hospital (entitled “The Place of Psychoanalysis in Medicine”), we
believe that the theory of the demand in the medical field laid
out in this lecture can be of help in spelling out the unconscious
determinant(s) at play in the reception of genetic information. Some of the literature partly addresses such unconscious
determinants upon the reception of medical information in a
Lacanian fashion, e.g., in the French-speaking psychoanalytic
tradition (Del Volgo, 1997; Brun, 2005 gathers important
collective proceedings on this topic; Lebrun, 2017; Weber, 2017). But we would like to approach them from an angle which, to
our knowledge, hasn’t been explored as such – especially in
genetics – that of the concept of demand1 (We leave aside non-
Lacanian approaches of demand in medicine; integrating them
would require a systematic review). In the 2000s, psychotherapists and family therapists were
already aware of the need to reflect upon the consequences of this
emerging state of affairs: “When we go for a routine physical, rather than making blanket
pronouncements about increasing exercise, lowering cholesterol,
and other preventive health measures, our physicians and nurse
practitioners are likely to draw individualized blueprints of
personal risk factors based on our specific personal genotype”
(McDaniel, 2005, p. 27). Thus, our goal will be to provide a presentation of the Lacanian
approach of demand, and to explore how it can be drawn upon
to understand the clinical stakes of pediatric genetics. 1While we stuck to the usual English translation, the meaning of the French
“demande” differs from that of the English “demand,” as will appear below in more
detail. While the English “demand” implies a positive requirement, and frequently
a dimension of command, the French “demande” (especially in its psychoanalytic
understanding) mostly refers to the expression of helplessness – so much so that it
often means to beg or to implore. INTRODUCTION As we
shall see, the interest of this specialty is that the unconscious
dynamics (aimed at by the notion of demand) implicitly at work
in the background of what is explicitly asked of the medical
practitioner, come more readily to the forefront: it is generally
parents who come for their child’s disease – this leads them
to express how they unconsciously represent their child. This
family context thus helps shed a strong light on the weight of the
unconscious fantasies at work in parental demand, which bear on
the psychical appropriation of the information and subsequent
decision-making. The question raised by this state of affairs is: what are
the personal, family and social effects of the possibility to
receive individualized medical recommendations based on
an unprecedented knowledge of one’s genetic and genomic
characteristics? To answer this question, social science research has explored at
length the personal and family effects of contemporary medicine
(cf. e.g., James et al., 2006; Hens et al., 2016), including the
indirect constraints embedded in genetic healthcare pathways
(e.g., Vailly, 2013). Within psychology, this question has been
scrutinized by cognitive-behavioral psychology (e.g., McDaniel,
2005) or systemic approaches, but few studies have addressed it
through the lens of psychoanalysis, with the exception of e.g.,
Feissel-Lebovici (2001), Aubert-Godard (2005), Driben (2011),
Gargiulo et al. (2017). Yet, the originality of psychoanalysis
lies in that it can spell out the unconscious determinants at
play in the reception of medical information (see e.g. Balint,
1957; Debray, 1996; Gutton and Raimbault, 1975; Raimbault,
1975; Sausse, 1997), of which genetic information is a subset. The specificity of a psychoanalytic approach to this question
lies in its grasp of how apparently remote autobiographical
elements and repressed childhood situations influence the very
thought processes of information understanding, by structuring
the individual’s personality up to the very way in which she
asks for help and assistance – and what she thereby genuinely
expects. In fact, the present paper presents a research trajectory, from
the experience of partaking in pediatric genetics consultations
within a renowned clinical genetics unit (Imagine Institute,
located at Necker Hospital in Paris), to the elaboration of a
funded research project on the psychosocial determinants of
the impact of genetic deafblindness (DéPsySurdi, see section
“Subjectivizing the Demand in Pediatric Genetics: Clinical
Practice and Research Perspectives”). Citation: November 2018 | Volume 9 | Article 2021 1 Frontiers in Psychology | www.frontiersin.org Lacanian Approach to Medical Demand Potier and Putois taking therapeutic decisions, from short-term life-or-death
transplant to long-term therapeutic compliance. THE MANIFEST
DOCTOR-PATIENT-FAMILY
RELATIONSHIP IN THE PEDIATRIC
GENETICS CONSULTATION This description of the pediatric genetics consultation derives
from OP and RP’s participation to routine clinical consultations
in the pediatric genetics unit of Necker-Enfants Malades Hospital
(Paris), and subsequent exchanges with medical practitioners in
the context of these consultations. In other words, material in this
section is not derived from research projects or investigations, but
from routine practice. After sequencing (biological analysis), another consultation
is planned for the announcement of the diagnosis. It is often
extremely emotional, due to the guilt-laden anticipation –
conscious or not – of having in fact transmitted the disease:
learning that the child indeed has a genetic disease would be
synonymous with having passed it on to him, news which can
sometimes trigger deferred psychotic or psychotic-like onsets –
be they momentary or revealing a personality structure –
if parents are fragile. (In de novo cases, where the child
is the first to have the disease because of a spontaneous
mutation in the parents’ sexual cells, we often witness guilt
as well, but in a reversed form, so to speak: parents feel
guilty because their child is the only one affected with the
disease.) p
In
France,
Necker
Hospital
has
always
been
at
the
forefront of an interdisciplinary dialog between medicine
and psychoanalysis – both in medical genetics and in child
psychiatry. At the time when Lacan examined “The Place
of Psychoanalysis in Medicine” (1966), one of his early
followers, Ginette Raimbault (M.D., Ph.D., psychoanalyst, who
introduced Balint groups in France along with her husband
Emile Raimbault) was head of an INSERM (French National
Institute for Mental Health) unit working on hereditary
child metabolic diseases. Since then, the interaction between
psychoanalysis and pediatric genetics at Necker has been
constant: many consultations are carried, on an ordinary basis,
by a pediatrician-geneticist and a psychoanalyst, who contributes
to the consultation as he sees fit (and can, if needed, meet with
patients afterward). A striking feature of such consultations is that, after the
practitioner has taken the time to announce the diagnosis,
and then given information about the transmission of the
disease (dominant vs. recessive, etc.) and the therapeutic and
lifecourse implications for the child, parents often have great
trouble making sense of the medical information they have
received – be it immediately or, more frequently, shortly
afterward. INTRODUCTION The methodological
constitution of this project is the result of the present work on
demand, which represents its preliminary stage in many respects. We first provide a description of the manifest doctor-patient
relationship in the pediatric genetics consultation, in order to
point where unconscious determinants can come to play a
role. We then develop Lacan’s understanding of the demand in
medicine – that is, in the patient–doctor relationship. We then
apply this understanding to pediatric genetics, by focusing on
what Raimbault, a pupil of Lacan’s, called “implicit demand”; and
we show how it this concept formed the starting point of the
aforementioned research project. Therefore, psychoanalysis can shed an original light on
two pressing issues which, albeit encountered daily in clinical
practice, are rarely dealt with directly in research papers,
especially outside of French-speaking psychoanalytic literature: (1) the unconscious determinants of patients’ difficulties to
understand genetic information; and (2) the unconscious determinants of subsequent attitudes
or
behavior
disregarding
(or
even
contradicting)
recommendations based on this information – e.g., in November 2018 | Volume 9 | Article 2021 Frontiers in Psychology | www.frontiersin.org 2 Lacanian Approach to Medical Demand Potier and Putois This is typically a three-step process:
first a clinical
examination (comprising the proposition to undergo genetic
sequencing and, in case of acceptance, the signature of an
informed consent form), followed by sequencing (genetic
analysis, on the basis of questions raised by the clinical
examination), and then – a couple weeks later – by the
announcement of the diagnosis (or lack thereof), along with
therapeutic advice (if possible). Our central idea is that Lacan’s understanding of the demand,
the genuine object of which isn’t medical information and/or
healing but knowledge of one’s fantasies about what takes place in
one’s body, allows for what we propose to call subjectivization –
that is, an awareness that the core object of one’s demand lies
elsewhere than in healing or care. Subjectivization accounts
for the apparent discrepancy between the information explicitly
received to the patient and his family, and their understanding
and subsequent actions. A specific trait of pediatric genetics is that clinical examination
involves questions regarding potential antecedents in family
history: the geneticist, in addition to undertaking a clinical
examination of the child and questioning his parents, searches
for signs of the disease in previous generations and relatives
while drawing a family tree. INTRODUCTION This entails that the explicit
parental demand to the practitioner directly puts parents
themselves in a position to receive confirmation that they
have transmitted the disease – if the genetic character of the
disease hasn’t been established already. This context cannot
but trigger family guilt: whatever the results of the analysis,
the anxiety to have passed on the disease is in everyone’s
minds – to the point, not infrequently, of inducing momentary
psychical splittings, as when parents, e.g., leave out of the family
tree a deceased relative who happened to have signs of the
disease. It is by taking into account this unconscious search for another
knowledge at work in the patient’s demand that the medical
practitioner will be in a position to both enable moments of
subjectivization, and deliver an adjusted medical response (both
in tone and in content) without being unknowingly caught in the
patient’s implicit demand. November 2018 | Volume 9 | Article 2021 The Subjectification of Jouissance:
Drive, Demand, and Desire In this context, jouissance refers to the untamed, not-yet-
organized circulation of excitation which takes place in the
infant’s body during the primordial interactions with his human
environment, whereby the infant experiences his body as such
(Lacan, 2016, Chap. 13). It is a pure erotic experience of one’s
organic being, in all its intensity – a jouissance of being (cf. also Dimitriadis, 2017) [It should be noted that while this
jouissance involves direct interactions with the Other as real,
since it corresponds to a “mythical” (Lacan, 2016, Chap. 13)
moment prior to the linguistic constitution of the subject qua
separated – more on this just below, the Other is correspondingly
not experienced as separated, but as part of a field of jouissance
comprising himself and the infant]. THE MANIFEST
DOCTOR-PATIENT-FAMILY
RELATIONSHIP IN THE PEDIATRIC
GENETICS CONSULTATION child – while it has just been made perfectly clear to them
that only symptom-oriented care (at best) could hereafter be
implemented. Geneticists experience the same type of perplexity during
follow-up consultations about medical decisions and care: often
do they see that the previously communicated (and repeated)
information
concerning
the
stakes
of
proper
therapeutic
decisions doesn’t seem to lead the parents to what would, from
the outside, appear as the most reasonable decision – such as
transplant, choice of medically adequate treatment, etc. For example, in pediatric immunogenetics, it is not rare to
see parents refusing life-saving bone-marrow transplants for their
children, because of the residual 10% risk of lethal outcome –
while, by refusing, they could be seen as in fact becoming
responsible for their children’s future death, bound to happen if
the immune system keeps deteriorating for genetic reasons. What the patient demands from the doctor as subject
supposed to know is a knowledge about the jouissance taking
place in his body. “The rapport thanks to which the doctor is
what he is, is the patient’s demand. Inside this strong relationship
where so many things take place, this dimension is fully revealed
in its original meaning (. . .): the relation to the body’s jouissance”
(Lacan, 1966, p. 309). How can a Lacanian approach to the patient–doctor
relationship taking place in pediatric genetics account for this
often paradoxical gap between the objective, medical information
transmitted to parents and patients, and its subjective reception
and elaboration? We first need to lay out Lacan’s understanding
of the demand in contemporary medical consultations (2). We
will then use these elements to explore how they come to play in
pediatric genetics (3). We thus need to briefly account for the constitution of the
subject’s relationship to the body’s jouissance, in order to shed
light on the patient’s demand to the doctor. THE MANIFEST
DOCTOR-PATIENT-FAMILY
RELATIONSHIP IN THE PEDIATRIC
GENETICS CONSULTATION Often do the geneticists find themselves in a position
to have to explain again the mode of transmission and its
implications, up to a point where it clearly appears that the
real question isn’t “what is the disease and how has it been
transmitted?,” but “Why us ?” – in other terms, an attempt
to make sense of blind biological fatality. The geneticist is the
bearer of bad news, his speech is very often received as an
oracle-like prediction (Feissel-Lebovici, 2001; Munnich, 2014);
yet, even when he has successfully isolated the pathogenic
variant, parents are often perplexed and cannot make sense
of these traumatic news. This is often evidenced in their
spontaneous question about what can now be done to cure their Classical
medical
genetics
is
mostly
concerned
with
Mendelian inheritance of pathogenic variants (along with
random spontaneous mutations, called de novo); as such,
it
mostly
focuses
on
monogenic
diseases
–
accounted
for by the variation of a single gene – or, at broadest,
on a defined set of genes. Pediatric genetics is thus the
best
setting
for
psychoanalytic
work
on
the
personal
impact
of
genetics:
since
it
revolves
around
Mendelian
transmission, its effects can be best witnessed in clinical
contexts where families come to the Medical Genetics Unit
to sort out both the name and the cause of their child’s
disease. November 2018 | Volume 9 | Article 2021 Frontiers in Psychology | www.frontiersin.org 3 Lacanian Approach to Medical Demand Potier and Putois “Cartesian dichotomy between thought and extension” (id.), as a
highly complex machine, in spite of the exponential development
of exploration and imaging devices which present a purified
version of it (cf. e.g., Potier, 2009). One should be aware that
this exponential development fostered an “epistemo-somatic rift”
(Lacan, 1966, p. 303) encouraging to (mis)understand the body
(soma, in greek) upon which medical knowledge (episteme)
should focus – and to miss that it is not to be understood as a
complex machine, but as a nexus of “jouissance” (id.). This rift is
typical of contemporary medicine: the diversity and complexity
of healing devices, machines and substances developed on the
basis of biomedical scientific progress tends to overshadow the
specific function of the practitioner, whose very authority and
personal prestige were deemed throughout the ages to be a central
part of his function (Lacan, 1966, p. 297). Frontiers in Psychology | www.frontiersin.org Consequences on the Patient–Doctor
Relationship: Subjectivizing the Demand The function of the Other’s initial response is thus to turn the
bodily jouissance of the cry into what Lacan calls an “invocative
drive” addressed to the Other (Lacan, 1973): by understanding
the cry as a call, the Other leads the infant to experience what
takes place in his body as a drive (with its source in a specific
erogenous zone, the mouth), aimed at satisfaction and expressed
as a demand. The cry thus becomes “the radical knot where
demand and drive come to be bound” (Lacan, 1973, session of
May 27th, 1964 – modified translation). It is for this reason that Lacan starts his conference on “The Place
of Psychoanalysis in Medicine” by stressing the “gap between
demand and desire” (Lacan, 1966, p. 302): while the manifest
demand addressed to the practitioner looks like a demand for
healing, the repressed signifiers of the desire of the Other to which
the demand can be related show the discrepancy between what he
demands and what he genuinely desires. At this level of primordial alienation, where the infant qua
subject of jouissance is bound to grasp what happens in his body
through the response of the Other, he undergoes an identification
to what Lacan calls object a (objet petit a) of the Other, wherein
he comes to wonder “what the Other wants from him” (Lacan,
2016) in so responding to his cry. When he is sent to the doctor, or comes to meet him, the
patient does not simply expects to be healed. He puts the
doctor to test, to see whether he can bring him out of
his condition; this is altogether different from healing the
patient, since this demand can imply that the latter very
much wants to remain ill. Sometimes the patient wants us
to authenticate his status of illness; in many other cases, his
obvious wish is that we help him remain ill, treat him in the
way he wants, which will help him remain settled within his
illness. I just need recall a recent experience: a patient, who
recently came in a formidable state of permanent anxious
depression having lasted for more than 20 years, was in utter
terror at the idea that I could do something for him. A Demand for Knowledge About
Jouissance of the Body In his remarks on “The Place of Psychoanalysis in Medicine”
(Lacan, 1966), Lacan writes that psychoanalysis can help medical
practice – and is, in this perspective, part of it – since it can spell
out what is at stake in the “authentically medical position” (Lacan,
1966, p. 301): namely, the mode of response to what the patient
unconsciously expects from the doctor, through what Lacan calls
“the demand” (id., p. 302). Paradoxical as it sounds, the patient’s doesn’t primarily expect
healing, which can be provided by therapeutic devices and agents
(surgery, drug, etc.). Aside from healing, “a certain something
remains constant, and every doctor knows what it is”: demand. And “the significance of the [patient’s] demand, wherein the
medical function authentically comes to play” (id., p. 302), is
that it is a “demand of knowledge.” That is, the demand to the
medical practitioner is an instance of what psychoanalysis calls
transference (Lacan, 1966, p. 308), whereby the subject supposes
a knowledge in the addressee of his demand, thereby considered
as a “subject supposed to know” (Lacan, 1973, Chap. 18; various
texts in Brun, 2005 refer to this point). At this mythical (i.e., reconstructed) stage of the constitution
of the subject, in the infant’s state of absolute dependence upon
its environment (Freud’s Hilfslosigkeit), it is the Other’s response
to the bodily manifestations of anxiety to which jouissance gives
rise which retroactively converts these manifestations into an
appeal. This is the first step of the process of subjectification
(subjectivation, Lacan, 2016, Chap. 12): the infant’s alienation to
the Other’s response. The paradigm case is the infant’s cry (cf. Lacan, 2016, Chap. 24): it is the “marks of [the Other’s] response that had the power
to turn his cry into a call” (Lacan, 2006, Remarks on Daniel
Lagache’s Presentation). While the infant’s cry doesn’t initially
express a specific need (since he wouldn’t know what he needs),
but instead manifests an unbearable excitation and is thus at the
level of jouissance, the Other (typically, the caregiver) interprets The object of this type of knowledge is not the body
defined as what can be “photographed, X-rayed, calibrated,
diagrammatized” and so on (Lacan, 1966, p. 303), by the medical
devices which help establish the diagnosis and heal. A Demand for Knowledge About
Jouissance of the Body In other
terms, the body is not to be understood, in the footsteps of the November 2018 | Volume 9 | Article 2021 4 Lacanian Approach to Medical Demand Potier and Putois it as a call for a specific action on his side – which will, in turn,
be determined by how He unconsciously represents the infant. This representation is constituted by signifiers, discrete elements
of speech considered as distinct sounds, independently of their
usual socially determined meaning; the specific signifiers which
constitute the Other’s representation of the infant will form the
latter’s ego-ideal, the very core of his subjectivity. infant, the maternal Other thus appears as desiring, since she
also cathects someone else, who partly accounts for what the
infant represents for her. Lacan calls this second step “separation”
(Lacan, 1973). Thus, at the end of this reconstructed two-step process of
unconscious subjectification by alienation/separation, the cry has
become a demand qua invocative drive. Correspondingly, its
object, i.e., what could genuinely satisfy it, isn’t merely the oral
partial object (breasts, etc.). Since the maternal Other, when
giving the breast to a crying infant, draws on the signifier-based
framework of Her representation of the infant qua object a of
desire, it is Her repressed representation of the infant qua object
a, which constitutes him as subject of the unconscious, which is the
object of his demand. These marks, in which the all-powerfulness of the response
are inscribed, are thus circled in reality with the signifier’s
line. It is not without reason that these realities are
called “insignias.” The term is nominative here. It is the
constellation of these insignias that constitutes the subject’s
ego-ideal (Lacan, 2006, Remarks on Daniel Lagache’s
Presentation). Thus, once the subject is constituted, everything that he comes
to voice will, from the perspective of the unconscious, have to be
understood as a demand, unknowingly articulating the signifiers
which constitute the coordinates of the particular object a that he
is for the Other. That is to say, the function of the Other’s response is to enable
a primary identification to bind the infant’s jouissance through
signifiers which represent him for the Other. Herein lies the “all-
powerfulness of the response.” Consequences on the Patient–Doctor
Relationship: Subjectivizing the Demand p
g
y
Therefore, he needs the Other to elucidate the signifiers of
primary identification (often written S1 by Lacan) by drawing
on a constellation of complementary signifiers (written S2) that
account for the Other’s choice of S1. Typically, S2 stands for the
Oedipal narrative which accounts for the unconscious choice of
S1 by the Other – most often the mother. [In most cases, the
maternal or mothering Other will be in a position to provide such
a constellation by drawing on the Name-of-the-Father, Lacan’s
formal re-writing of the Oedipal complex (Lacan, 1998); for a
more detailed recent presentation, cf. e.g., Razon et al., 2017, see
section “The Manifest Doctor-Patient-Family Relationship in the
Pediatric Genetics Consultation.”) In such a second step, whereby
the primordial Other is divided by the necessity to account for his
choice (most often by leaving room in the S2 for another figure
co-defining the infant’s identity through a paternal function, such
as the father), the object a to which the infant was identified
acquires a new meaning through S2 – and the infant can thus
know what he is for this Other, i.e., what the Other wants from
him (Lacan, 1973, 2016). From the perspective of the Other,
the infant becomes an object of desire since he is viewed as a
representative of another desired figure; he becomes, as Lacan
puts it, “phallicized” (Lacan, 1973). From the perspective of the (. . .) As soon as we’ve pointed out [the gap between demand
and desire], it appears that it isn’t necessary to be a psychoanalyst,
nor even a doctor, to know that once anyone, be they our best
friend, male or female, demands something, it is in no way
identical to – and, sometimes, in full opposition with – what they
desire (Lacan, 1966, p. 302). What the patient desires can thus, depending on the
structure of the signifiers which constitute him as subject of
the unconscious, amount to various types of relationship with
the Other – such as, e.g., remain dependent from Him (“help
him remain ill”) – which are then projected onto the person
of the medical practitioner. An Instance of a Setting Enabling
Subjectivization: The Instant to Say We can illustrate this concept by commenting an example
through which Del Volgo presents the original clinical setting
that she calls the “instant to say” (1997, p. 61), which we view
as a typical setting enabling subjectivization. Del Volgo, both a
hospital medical practitioner and a Lacanian psychoanalyst, gives
examples of how, within the context of a medical consultation,
she asks patients about their medical history in such a way as
to enable an “instant to say.” This refers to a logical moment
when patients, by recalling the important events of their life in
the course of recounting the history of their illness and its various
stages or occurrences, are presented with the opportunity to grasp
the signifiers with which they describe the illness in relation to
important prior life events. While this opportunity isn’t presented
explicitly, or as a goal of the consultation, this associative process
opens up a space aside the healing-oriented dimension of the
medical response, and gives them a chance to grasp and question
the meaning of their medical demand – that is, the structure given
to their jouissance by prior important life events. In so doing, she
doesn’t respond do the immediate demand but tries to help the
patient gradually become aware of the subjective significance of
his symptoms, i.e., of the fantasy which underlies them. This analysis of the medical function thus implies that it
depends on the doctor to hear the patient’s demand as the
manifestation of a desire to know something about his jouissance:
he can thereby help the patient become aware of his desire,
instead of responding to the demand solely by drawing on the
position granted to him by his knowledge and position. This is
certainly not to say that the medical practitioner has to explicitly
interpret the patient’s discourse: a medical consultation isn’t a
preliminary interview prior to the initiation of a psychoanalytic
cure. But being aware that the demand’s object is knowledge upon
the patient’s jouissance can help make the latter aware that the
truth of his demand (in the psychoanalytic sense of the term:
the subjective truth) doesn’t primarily lie in medical knowledge –
once again, a preliminary step to an adjusted medical response in
terms of cure and healing. We will comment on a case that she presents in Del Volgo
(1997). Consequences on the Patient–Doctor
Relationship: Subjectivizing the Demand These types of relationship with
the Other refer to the type of object a to which the subject is November 2018 | Volume 9 | Article 2021 Frontiers in Psychology | www.frontiersin.org 5 Lacanian Approach to Medical Demand Potier and Putois We propose to call the awareness that the medical practitioner
can help the patient experience a subjectivization of the latter’s
demand. Subjectification, the word aptly chosen by A. Price to
translate the French word “subjectivation” (Lacan, 2016, Chap. 12), refers to the constitution of the subject through alienation
to, and separation from, the Other; we view subjectivization as
referring to something different, namely the process of becoming
aware of the essentially subjective nature of the demand to the
practitioner concerning what happens in his body. Subjectivizing
means understanding, to some extent, that the meaning of
this demand derives from elements of one’s own subjective
coordinates; in Lacanian terms, this amounts to understanding
that the signifiers of one’s demand have to be referred to the
primary signifiers in the Other, which assign the subject to a
certain position qua object a of the desire of the Other. A medical
consultation carried by a practitioner aware of both medical
stakes and the subjective meaning of demand, can help the patient
partially grasp this subjective meaning, and question what it is he
wants from the practitioner. reduced by the desire of the Other – this is the formula of the
fundamental fantasy (Lacan, 1973), which formalizes the role and
the organ (mouth, etc.) to which the subject identified at the step
of separation from the Other. It is this formula, to which the
subject identified in separation, which gives its shape to the desire
of the Other, and that the subject unknowingly seeks to uncover
by voicing his demand, which is at bottom transference, i.e., a
“demand of knowledge” (Lacan, 1966, p. 308). reduced by the desire of the Other – this is the formula of the
fundamental fantasy (Lacan, 1973), which formalizes the role and
the organ (mouth, etc.) to which the subject identified at the step
of separation from the Other. It is this formula, to which the
subject identified in separation, which gives its shape to the desire
of the Other, and that the subject unknowingly seeks to uncover
by voicing his demand, which is at bottom transference, i.e., a
“demand of knowledge” (Lacan, 1966, p. 308). Consequences on the Patient–Doctor
Relationship: Subjectivizing the Demand Hence the importance of the medical response: strictly
understanding what the patient says as a demand for healing via
a cure, and thereby missing that the signifiers used or hinted at by
the patient are indirectly referring to something else (the object
a) will prevent the doctor from grasping that what he wishes is to
know the truth about the structure given to his jouissance by the
desire of the Other, i.e., about the fantasy at play. Correspondingly, it is by taking into account this unconscious
search for another knowledge at work in the patient’s demand
that the medical practitioner will be in a position to deliver
an adjusted medical response (both in tone and in content). In the medical consultation, especially in the context of heavy
medical examinations, leaving out this dimension will typically
lead the patient to persist in fulfilling his unconscious role in
the fantasy (e.g., request more and more examinations, or act
in opposition with what he is told). Reversely, the medical
consultation (as Del Volgo, 1997 has insisted) provides the
practitioner with a context propitious to help the patient gain
awareness, and question the consistency, of the knowledge of
his jouissance that he supposes that the Other holds – in a
movement analogous to the end of a psychoanalytic cure, where
transference is dissolved, i.e., the consistency of the subject
supposed to know collapses (Lacan, 1968(unpublished), Session
of January 10th, 1968). “On the one hand, [the doctor] deals
with an energetic cathexis, the potency of which he cannot
suspect if he isn’t told about it” – i.e., transference – and
“on the other, he needs to put this cathexis between brackets
precisely because of the power that he possesses, that he needs to
distribute [i.e., medicine, OP], and of the scientific plane within
which he is situated” (Lacan, 1966, p. 308). In so doing, he
puts his medical knowledge between brackets in order to gain
access to the patient’s representation of his knowledge about
jouissance, in order to be able to provide the right, adjusted medical
response. Consequences on the Patient–Doctor
Relationship: Subjectivizing the Demand Focused on producing in the subject an interrogation on the
genuine meaning of his demand (and open up the way for a
potential further inquiry on this desire itself), subjectivization in
a medical context is a the condition for adjusted medical action,
and a potential preliminary step with respect to a potential deeper
elucidation – such as the one carried in a psychoanalytic cure,
which ultimately aims at helping the subject move beyond his
assignation as object a of the Other’s desire. Frontiers in Psychology | www.frontiersin.org November 2018 | Volume 9 | Article 2021 An Instance of a Setting Enabling
Subjectivization: The Instant to Say An elderly asthmatic patient, Ange, experienced a severe
asthma crisis upon learning from the specialist that his wife,
after 3 weeks of nocturnal hallucinations which made him feel
“lost” much like an orphan, was in fact not suffering from November 2018 | Volume 9 | Article 2021 6 Lacanian Approach to Medical Demand Potier and Putois a brain tumor, as initially suspected. Being asked to recall
important elements in his life, he indicates that he has been
repeatedly and unexpectedly been put in the position to be the
closest to his mother: his father died in the beginning of World
War II, when his older brothers had already left the house. He experienced this as becoming the man in the house – an
important signifier for his personal history. His first respiratory
crisis occurred at age 30, “the age of adulthood” (where he could
go see a doctor, unlike childhood where he was once beat up for
doing so): he accidentally started spitting blood during physical
effort, which (he says) includes physical intimacy. It thus seems
that respiratory problems became associated with fantasies of
castration as a punishment for Oedipal desire, summoned (in
accordance with Freud’s bi-phasic trauma theory) in the context
of adulthood and conjugal life. It is as if the guilt of desire
(being put in the position of a phallicized object a vis-à-vis the
Other in the fantasy) could find a somatic expression – castration
symbolized as bleeding out during effort; and that, conversely,
the presence of the Other was experienced as the approach of
a forbidden oral object a, thus causing in his body a symbolic
equivalent of castration through hysterical conversion. this is that the demand for diagnosis and cure is voiced for
the child by the parents – the unconscious of whom largely
contributed to structuring the child’s – who feel responsible for
his disease since it is viewed as hereditary (at least potentially: the
cause is sought for in previous generations). An Instance of a Setting Enabling
Subjectivization: The Instant to Say “the child’s disease thus seems to reveal the family’s problem and
its singular drama, which is actualized in the disease and feeds off
of it, but isn’t properly speaking caused by it. The difficulties faced
by doctors partly stem from the fact that they only hear the explicit
demand (‘Cure this crisis!’) and not the implicit one (‘This is our
drama’)” (Raimbault in Lacan, 1966, p. 313). Any medical discourse concerning this hereditary agent will
thus be filtered by a pre-existing family fantasy organizing what
she calls implicit demand to the practitioner. We now draw on this conceptualization of the demand as
carrying a repressed desire open to subjectivization (and open to
further elucidation), and we turn to pediatric genetics. The specificity of the notion of implicit demand is that it
refers to the parents’ quest for help with respect to a guilt
which, albeit coming to the forefront at the occasion of the
child’s disease, predates it. Raimbault’s main clinical finding
is thus that the disease is filtered by the “window of the
fantasy”: to put it in the Lacanian framework which underlies
her work, the disease is experienced by the parents (especially the
mother) as a punishment for their normal anticipated fantasmatic
elaboration of the status of the child qua object a, prior to
any medical condition. Since this anticipated elaboration – way
before the birth of the child – cannot but include an element of
repressed guilt (even in neurotic contexts: a child is always partly
viewed by both parents as an Oedipal child), the subsequent
disease is experienced, through an unconscious displacement, as
punishment for the accomplishment of the Oedipal wish to have
a child with one’s parent. An Instance of a Setting Enabling
Subjectivization: The Instant to Say As we mentioned above, the main proponent of applying
Lacan’s theory of the medical demand to pediatric genetics was
Ginette Raimbault, in charge of research on the unconscious
stakes of medical consultation at INSERM (French National
Institute for Mental Health), and whose clinical field was a
pediatrics unit working on hereditary child metabolic diseases –
the precise hereditary cause of which was largely unknown
at the time, for lack of adequate sequencing apparatus and
knowledge. In 1966, right after Lacan gave his lecture on
“The place of Psychoanalysis in Medicine,” she gave a didactic
presentation of her research – which consisted in assisting silently
to consultations and elaborating on the unconscious dynamics
at stake in the family’s demand. This is how she describes these
dynamics: “As early as during the first interview with the medical
practitioner, the parents formulate the results of their own
research about the etiology of the disease, considered as a trouble. (. . .) The parents’ formulation shifts from ‘this makes no sense’
to ‘this is the sense we give to this disease”’ (Raimbault in Lacan,
1966, p. 313). This interpretation was confirmed through transference
during the next consultation, when he mentioned that a cardiac
accident occurred while he was eating sweets on the anniversary
day of their first consultation: the reminiscence of the first
consultation during the second one, and the structure of the
Oedipal fantasy within which he is caught up, accounts for
the symbolic equivalence between the forbidden pleasure of
eating sweets and becoming intimately close with the mother of
childhood. The cardiac accident is thus a transferential replica
of his first respiratory problems at age 30, confirming that these
series of bodily events can be understood against the background
of the way in which his jouissance is structured – namely,
through an oral Oedipal fantasy. Those elements constitute the
background on which the patient’s associations, supported by
Del Volgo’s psychoanalytic listening, shed a partial light during
this sequence; it was then up to the patient to subjectivize the
connection between these past events and the actual occurrences
of respiratory problems. While the subjective sense given by the family to the
disease partly depends on the medical antecedents, the lack of
information or the powerlessness of medical science (op. cit.), it
largely derives from “the elaboration of fantasies concerning the
agent of the disease” (Raimbault in Lacan, 1966, p. 313). November 2018 | Volume 9 | Article 2021 Explicit vs. Implicit Demand in Clinical
Practice As mentioned above, pediatric genetics is particularly interesting
to study the demand at work in contemporary medical practice
since the structure of the fantasy which organizes the patient’s
desire (and thereby filters the reception of information) is often
more readily accessible during the consultation. The reason for November 2018 | Volume 9 | Article 2021 Frontiers in Psychology | www.frontiersin.org 7 Lacanian Approach to Medical Demand Potier and Putois somewhere else. She replied (thereby illustrating the equivocation
enabled by the signifier “guilt”): “what do you mean? I am
by no means irresponsible! I’m doing my best here!” Her
mastery of French language was more than sufficient to rule
out a cognitive explanation for her apparent mistake. In so
responding, she showed us how guilty she does feel for their
disease, experienced transitively as a punishment for what (in
the rest of the consultation, in relation to biographical elements)
most likely appeared to be the structurally normal (see section
“The Subjectification of Jouissance: Drive, Demand, and Desire”),
predating Oedipal fantasy of receiving a child from her father –
the paradigm forbidden desire of which, at a certain level,
she unconsciously expected the consultation to relieve her, by
helping her formulate it. The singular drama was thus that she
unconsciously experienced this forbidden desire, upon which
becoming a mother largely relies, as directly punished by the
disease. It is this unconscious connection, qualifying her relation
to her children qua phallic objects a (because of the Oedipal
structure of the Other organizing her unconscious), that she
needed to subjectivize; for ultimately, the way to partly soothe
this guilt is to start by acknowledging it, which is the object of
her implicit demand to medical knowledge about what takes place
within her children’s bodies, and therefore filters how she heard
OP’s intervention. The notion of implicit demand thus directly echoes Lacan’s
characterization of transference on the doctor as a demand
for knowledge upon one’s jouissance, and narrows it down to
the context of hereditary diseases: what is implicitly demanded
is knowledge about the family fantasy giving shape to their
guilt. An Example of Implicit Demand: A
Consultation in Pediatric Genetics The medical context in which hereditary child diseases nowadays
take place is pediatric genetics, wherein such implicit demand
unfolds. The following example illustrates elements present
within a host of consultations, and comes from OP’s practice
of pediatrician-genetician/psychoanalyst dual consultations in
pediatric genetics. Only de-identified data were used; therefore,
an ethics approval was not required for the use of this material
as per the Institution’s guidelines and national regulations. It
shows how the explicit demand carries an implicit one, which
filters both the reception of information and the subsequent
decision-making of patients. Unfortunately, this subjectivization (realizing the relation
between her experience of the disease and a guilt of a
different origin) was made extremely difficult by the pressing
therapeutic context, where a decision had to be made in the
near future concerning the bone marrow transplant. In other
words, aside the response which she unconsciously sought
concerning the fantasmatic cause of what was taking place
within their children’s bodies, a healing-oriented response also
had to be given her concerning the stakes and urgency of the
transplant. Upon hearing about the necessity to soon make a
decision concerning this matter, she said she was extremely
reluctant to accept it, because of the residual 10% chance of
lethal outcome (in spite of the certainty of such an outcome
in the absence of transplant). One can wonder whether a
masochistic need to be punished for the Oedipal character of
her fantasy, which enabled her to represent her children as
phallicized objects a in the first place, could account for her
decision: wouldn’t a lethal amount (inexorable in the absence
of transplant) symbolically amount to a paradigm punishment
for her forbidden fantasy? In this case, what appeared to be the
structure of her fantasy could account for her fantasy, with its
masochistic components. Her behavior, seemingly paradoxical
with respect to the perspective of healing and cure, thus appears
in a new light (see section “Consequences on the Patient–Doctor
Relationship: Subjectivizing the Demand”). In this context, that of the pediatrician-genetician and
psychoanalyst, the difference with both Del Volgo’s setting and
Raimbault’s research is that the psychoanalytic perspective is
embodied by a specific person (not the doctor), who also actively
partakes in the consultation, sometimes to an important extent –
when the weight of the implicit demand comes to the forefront. Explicit vs. Implicit Demand in Clinical
Practice It is in this wake that Raimbault insists that what
matters most, on the side of medical practitioners, is to prevent
stereotyped attitudes and responses based on unquestioned
personal assumptions concerning what stands as appropriate
behavior in those medical situations: they are laden with
the practitioners’ personal subjective organization, and would
prevent him from grasping the family’s implicit demand
(Raimbault in Lacan, 1966, p. 314). The core of the knowledge that he is unconsciously asked by
the family – the object a of the family’s demand, so to speak (and it
most often is the mother’s, in these circumstances) – concerns the
particular structure of the desire of having a child, of which they
(unavoidably) feel guilty. Responding to their implicit demand
would amount to help them subjectivize this family fantasy. An Example of Implicit Demand: A
Consultation in Pediatric Genetics It is not only listening, but also active interventions, which open
up a space of subjectivization, i.e., of relating the signifiers of
the demand (the explicit demand, in Raimbault’s quote) to those
of the underlying fantasy of the implicit demand of the family
singular drama. This is sometimes needed in order to shed light
on the extent to which this demand filters medical information
and subsequent behavior. The following example is reduced to a few elements
for anonymity reasons. A young mother of two adolescents
was extremely reluctant to try a bone marrow transplant
which could save them both of a rapidly developing disease
enabled by a hereditary recessive immunodeficiency. Hearing
the unmentioned guilt present in her speech, OP told her
“in any case Madam, you are not responsible for your sons’
disease” – in order to stress that she couldn’t know, medically
speaking, that mothering them would lead to transmitting them
the disease, but that her apparent sorrow might be rooted Frontiers in Psychology | www.frontiersin.org Demand-Based Starting Point of a
Qualitative Research: The Subjective
Effects of Genetic Deafblindness Finally, we would like to give a brief illustration of the demand-
based rationale of an ongoing qualitative research based on
this conception of the demand in pediatric genetics. This November 2018 | Volume 9 | Article 2021 8 Lacanian Approach to Medical Demand Potier and Putois multidisciplinary research focuses on the subjective effects of
genetic deafblindness on autonomy in child, adolescent and adult
patients with Usher, Wolfram and Stickler syndromes (research
codename: DéPsySurdi). It is funded by the French Rare Disease
Foundation (“Fondation Maladies Rares”); RP is its principal
investigator, and it has been made possible by a close partnership
with the Reference Center for Genetic Deafness (INSERM –
U587, dir. Dr. S. Marlin). the degree to which the child’s participation to the family
fantasy – and conversely, his degree of autonomy with respect
to it. What room do they leave for the gap between their child’s
autonomy in the expression of his demand, and their own
representation of him (laden with the guilt of his syndrome,
which clouds the structural predating guilt)? And in cases where
this gap is thin, is his demand devoid of singular desire, i.e.,
just a reflection of his main caregiver’s fantasy, in which he
would be caught up? Or does it present aspects of a singular
desire of separation qua phallicized object a, waiting to be
acknowledged as such? This stake is particularly crucial, since the
context of deafblindness leaves less room for separation, since
communicating often requires to use tactile sign language – and
thus to touch. What of the potential equivocity of the signifier in
these contexts? What we briefly present here is the nucleus of the
psychoanalytic rationale of this research, jointly conceived by RP
and OP on the basis of a Lacanian approach to the demand in
the context of genetics (and in particular pediatric genetics). This
nucleus is both the result of a research on medical demand with
a focus on pediatric genetics, and the basis of the specifically
psychoanalytical contribution to the DéPsySurdi project; on this
basis, collaborators in psychology, medicine and social science
joined in order to turn this nucleus into an exploration of the
effects of genetic deafblindness at a psychosocial level. CONCLUSION It is
for this reason what we chose to focus on pediatric genetics, since
this clinical field is both saturated with such projections, but at
the same time open to potentially disalienating interventions –
especially if children themselves, as well as other members
of the family, actively partake in the medical exchanges, so
as to distinguish their own speech to their parent’s implicit
demand. This
initial
context
raises
specifically
psychoanalytic
questions: doesn’t the variety of available supports sometimes
cloud the subjective significance of the syndromes? In other
words, behind the need for help and support, does the current
available medico-social leave room for subjectivization in the
families’ and patients discourse on the handicap? To what
extent can they question the signifiers which, at the manifest
level, they use to refer to the everyday impact of the handicap,
and ways to alleviate its burden? This would mean having the
opportunity to relate the gene-based loss of autonomy (along
with the parental guilt which accompanies it and is reinforced
by genetic sequencing) to what Raimbault called the predating
family fantasy which gives its particular sense to their child’s
handicap. In this respect, a particularly sensitive question is Demand-Based Starting Point of a
Qualitative Research: The Subjective
Effects of Genetic Deafblindness (Our
methodology, which we cannot fully unfold here, relies on semi-
structured interviews using sign language or tactile sign language,
in order to leave as much room as possible for association,
and more generally punctual emergences of formations of the
unconscious.) These are the questions which led us to decide to focus on this
cluster of syndroms, in order to help doctors position themselves
with respect to the unconscious question concerning one’s body
at play in the demands for support that they receive. CONCLUSION The gene-based Usher, Wolfram and Stickler syndromes
gradually affect both hearing and sight up to partial or total
auditory and visual deficits, resulting in deafblindness (a specific
handicap, wherein large parts of audio-visual compensation is
impossible). The effects of this handicap on one’s autonomy
appear to vary greatly; it has important psychiatric comorbidities,
such as depression due to increased social isolation. We decided
to examine the effects of deafblindness on autonomy in child,
adolescent and adult subjects because autonomy is centrally
impacted by this handicap, and is thus the natural manifest
object of family and patient demand: the demand for medical
and social help and support greatly focuses on compensating this
handicap, especially in parents with children and teens affected
with these syndromes. Therefore, various strategies, devices (e.g.,
technological) and personalized supports (personal or family
assistants, etc.) are devoted to this compensation, and thereby
help young patients and their parents achieve social participation
and self-realization: the explicit objects of the families’ demands
are means to ease the burden of the handicap and facilitate
interaction. In this paper, we wanted to draw on Lacan’s take on the demand
to stress that the exponential development of personalized and
stratified medicine, which help provide previously unexpected
adjusted cure and healing, requires medical practitioners to
remain sensitive to the dimension of the demand for knowledge
about jouissance, in order to prevent their response (cure,
investigations, etc.) from reinforcing the underlying repressed
fantasies, with their masochistic basis. It is even more important when the object of this knowledge
is what takes place in someone else’s body: these situations are
often quite projective, in the sense that it is difficult for caregivers
to acknowledge that their understanding of their child’s bodily
symptoms is heavily influenced by his role in their fantasy. This
is particularly true when the child represents the object of the
Mother’s (and not the couple’s) fantasy, as Lacan wrote in the first
“Note on the child” in 1969 (Lacan, 1986). The unconscious guilt of having transmitted the disease, even
in cases where transmission cannot be established (which are
numerous, even as of today), contributes to these projections. Frontiers in Psychology | www.frontiersin.org November 2018 | Volume 9 | Article 2021 REFERENCES Lacan, J. (1986). Notes Sur L’enfant (1969). Ornicar 37, 13–14. [English Transl. by
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Seuil. Lacan, J. (2006). Ecrits: The First COMPLETE Edition in English, eds B. H. Fink and
R. Grigg (New York, NY: Norton & Company). Balint, M. (1957). The Doctor, His Patient and the Illness. London: Churchill
Livingstone. Brun, D. (2005). “Violence de l’annonce, violence du dire,” in Proceedings of the
7ème Colloque de l’Association Médecine et Psychanalyse, eds D. Brun (dir.) and
É. Freudiennes. Lacan, J. (2016). The Seminar, book X: Anxiety (1962-1963), tr. A. Price. Cambridge:
Polity Press. Lebrun, J.-P. (2017). De la maladie au malade. Psychanalyse et medicine
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017.02 Christenhusz, G. M., Devriendt, K., and Dierickx, K. (2013). Disclosing incidental
findings in genetics contexts: a review of the empirical ethical research. Eur. J. Christenhusz, G. M., Devriendt, K., and Dierickx, K. (2013). Disclosing incidental
findings in genetics contexts: a review of the empirical ethical research. Eur. J. Med. Genet. 56, 529–540. doi: 10.1016/j.ejmg.2013.08.006 Med. Genet. 56, 529–540. doi: 10.1016/j.ejmg.2013.08.006 McDaniel, S. (2005). The Psychotherapy of genetics. Fam. Proc. 44, 25–44. doi: 10.1111/j.1545-5300.2005.00040.x Debray, R. (1996). Clinique de l’expression somatique : psychanalyse des liens
psyché-soma. Lausanne: Delachaux et Niestlé. Munnich, A. (2014). La génétique est-elle inhumaine? Esprit 7, 66–74. doi: 10.3917/
espri.1407.0066 Del Volgo, M.-J. (1997). L’instant de dire : le mythe individuel du malade dans la
médecine moderne. Erès: Ramonville Saint-Agne. Potier, R. (2009). L’image en médecine, esquisse et précipice. Res. Psychoanalys. Recherches en Psychanalyse 8, 164–169. Dimitriadis, Y. (2017). The psychoanalytic concept of jouissance and the kindling
hypothesis. Front. Psychoanal. 8:1593. doi: 10.3389/fpsyg.2017.01593 Raimbault, G. (1975). L’enfant et la mort: problèmes de la clinique du deuil. Privat:
Dunod. Driben, A. (2011). Les filles et leur père. Champ Psy. 6, 51–62. doi: 10.3917/cpsy. 060.0051 Razon, L., Putois, O., and Vanier, A. (2017). The lacanian concept of cut in light of
lacan’s interactions with maud mannoni. Front. Psychol. 8:2177. doi: 10.3389/
fpsyg.2017.02177 Feissel-Lebovici, A. (2001). Le gène et son génie. Patient, médecin et psychanalyste
face à l’hérédité et au cancer. Erès: Ramonville Saint-Agne. Gargiulo, M., Tezenas du Montcel, S., Jutras, M. FUNDING psychoanalytic basis of the deafblindness research, and is the
Principal Investigator of the resulting psychosocial project
(DéPsySurdi – http://www.depsysurdi.fr/). He has given useful
comments on a previous draft of this paper, and has agreed
to the final version. OP has conceived the structure of the
paper and its argumentation (topic and organization were
based on his post-doctoral research), taken part in most of
the consultations mentioned in the paper, has co-conceived the
psychoanalytic basis of the deafblindness research, and written
the paper. This research received funding through a grant awarded to the
project “DéPsySurdi” (PI: RP) by the Fondation Maladies Rares’
Social and Human Sciences program. This research received funding through a grant awarded to the
project “DéPsySurdi” (PI: RP) by the Fondation Maladies Rares’
Social and Human Sciences program. OP’s post-doctoral fellowship was supported by the “Who Am
I?” Laboratory of Excellence #ANR-11-LABX-0071 funded by the
French Government through its “Investments for the Future”
program operated by the ANR under grant #ANR-11-IDEX-
0005-01. AUTHOR CONTRIBUTIONS RP has contributed to part of the material (including clinical)
presented in this paper through an uninterrupted dialog
with OP during the latter’s post-doctoral research (which
he de facto partly supervised); he has co-conceived the November 2018 | Volume 9 | Article 2021 9 Lacanian Approach to Medical Demand Potier and Putois November 2018 | Volume 9 | Article 2021 REFERENCES F., Herson, A., Cazeneuve, C., and Sausse, S. (1997). Le miroir brisé. L’enfant handicapé, sa famille et le psychanalyste. Paris: Calmann-Lévy. Gargiulo, M., Tezenas du Montcel, S., Jutras, M. F., Herson, A., Cazeneuve, C., and
Durr, A. (2017). A liminal stage after predictive testing for Huntington disease. J. Med. Genet. 54, 511–520. doi: 10.1136/jmedgenet-2016-104199 Durr, A. (2017). A liminal stage after predictive testing for Huntington disease. J. Med. Genet. 54, 511–520. doi: 10.1136/jmedgenet-2016-104199 Vailly, J. (2013). The birth of a genetics policy. Social issues of newborn screening. New York, NY: Routledge. Gutton, P., and Raimbault, G. (1975). L’enfant devant la maladie somatique: 11
textes. Paris: SPEI. Weber, J.-C. (2017). La consultation. Paris: PUF. Weber, J.-C. (2017). La consultation. Paris: PUF. Hens, K., Peeters, H., and Dierickx, K. (2016). Genetic testing and counseling in
the case of an autism diagnosis: a caregivers perspective. Eur. J. Med. Genet. 59,
452–458. doi: 10.1016/j.ejmg.2016.08.007 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. James, C. A., Hadley, D. W., Holtzman, N. A., and Winkelstein, J. A. (2006). How
does the mode of inheritance of a genetic condition influence families? A study
of guilt, blame, stigma, and understanding of inheritance and reproductive
risks in families with X-linked and autosomal recessive diseases. Genet. Med. 8, 234–242. doi: 10.1097/01.gim.0000215177.28010.6e Copyright © 2018 Potier and Putois. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these
terms. Lacan, J. (1966). “La place de la psychanalyse dans la médecine,” in Psychanalyse
des enfants séparés (2010), ed. J. Aubry (Paris: Flammarion). Lacan, J. (1973). The Seminar 1964, Book XI, The Four Fundamental Concepts of
Psycho-Analysis [1973]. New York, NY: Karnac. November 2018 | Volume 9 | Article 2021 Frontiers in Psychology | www.frontiersin.org 10
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Nevus of Ota – an intraoral presentation: a case report
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CASE REPORT Open Access Maguire and Holt Journal of Medical Case Reports (2019) 13:174
https://doi.org/10.1186/s13256-019-2101-0 Maguire and Holt Journal of Medical Case Reports (2019) 13:174
https://doi.org/10.1186/s13256-019-2101-0 Background the left buccal mucosa. Her past medical history revealed
a diagnosis of “oculodermal nevus.” She recalled having
pigmentation in her left eye from birth and pigmentation
of skin of the left face since the age of 13 years, for
which she received laser treatment for cosmetic pur-
poses. The patient also reported annual monitoring of a
benign intracranial tumor along with close monitoring
by ophthalmology and dermatology divisions. She did
not take any regular medications. She did not smoke or
consume alcohol. She is single and lives on her own with
no dependents. She lives close to her mother, who
attended the appointments with her. Nevus of Ota or “oculodermal melanocytosis” is a rare
congenital hamartoma of dermal melanocytes causing a
blue-gray hyperpigmentation of the eye and surrounding
structures [1]. Originally described by Ota and Tanino in
1939, it mainly affects the ophthalmic and maxillary di-
visions of the trigeminal nerve and is most prevalent in
the Japanese population, with an incidence reported be-
tween 0.2% and 1% [2]. Oral involvement of the nevus of
Ota is very rare [3]. We describe the first reported case
of unilateral oculodermal melanocytosis with oral buccal
mucosal involvement in a Caucasian woman. It is im-
portant to be aware that nevus of Ota can present orally. On examination, a subtle but diffusely speckled bluish
pigmentation was observed to the left midface involving
the infraorbital and zygomatic regions. A post–laser
therapy yellow hue was noted on the left periorbital skin. Pigmentation of the sclera and conjunctiva was also
observed. Intraorally, an inhomogeneous, blue-gray, dif-
fuse hyperpigmentation affecting the entire left buccal
mucosa was noted. Mild pigmentation of the left hard
palate was also noted. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Nevus of Ota or “oculodermal melanocytosis” is a rare congenital hamartoma of dermal melanocytes
causing a blue-gray hyperpigmentation of the eye and surrounding structures. The condition, originally described
by Ota and Tanino in 1939, mainly affects the ophthalmic and maxillary divisions of the trigeminal nerve. We
describe the first reported case of unilateral oculodermal melanocytosis in a Caucasian woman with oral buccal
mucosal involvement. Oral involvement of nevus of Ota is very rare. Case presentation: A 48-year-old Caucasian woman was referred by the dermatology division to the oral medicine
department at the University of Liverpool School of Dentistry with new-onset oral pigmentation to the left buccal
mucosa. The patient had a previous diagnosis of oculodermal nevus. Conclusion: An incisional biopsy of the left buccal mucosa was completed. The report stated that histological and
immunohistochemical features were in keeping with a blue nevus, but within the context of the preexisting
occulodermal pigmentation, a diagnosis of oculodermal melanocytosis, also known as “nevus of Ota,” was made. The patient will be kept under review in the oral medicine department because the progression of the lesion on
the left buccal mucosa requires active monitoring owing to the potential for malignant change. The patient also
requires regular review in the dermatology and ophthalmology divisions. Keywords: Oral, Pigmentation, Nevus, Ota, Oculodermal, Buccal, Mucosa Nevus of Ota – an intraoral presentation: a
case report Jennifer Maguire1* and Deborah Holt2 Case presentation A 48-year-old Caucasian woman was referred to the oral
medicine department at the University of Liverpool
School of Dentistry with new-onset oral pigmentation to * Correspondence: jenmag48@hotmail.co.uk; jennifer.maguire@olchc.ie
1Our Lady’s Children’s Hospital Crumlin, Dublin, Ireland
Full list of author information is available at the end of the article cle is distributed under the terms of the Creative Commons Attribution 4.0 Competing interests The prevalence of this condition is greatest in the
Asian continent, affecting up to 1% of the Japanese
population [2]. One study has reported an incidence of
0.038% in Caucasians, although this is poorly docu-
mented [7]. About 85% cases of nevus of Ota occur in
females. The authors declare that they have no competing interests. Received: 9 January 2019 Accepted: 28 April 2019 Received: 9 January 2019 Accepted: 28 April 2019 Ethics approval and consent to participate Ethics approval and consent to participate
Not applicable. Not applicable. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Consent for publication Written informed consent was obtained from the patient for publication
of this case report and any accompanying images. A copy of the
written consent is available for review by the Editor-in-Chief of this
journal. A literature search of PubMed and Embase revealed
only two cases (in India) of nevus of Ota affecting the
buccal mucosa [5, 6]. Intraoral presentations more fre-
quently involve the palatal mucosa. Author details
1 Use of lasers for treatment of nevus of Ota has be-
come helpful in the management of dermal nevi. Cur-
rently, Q-switched lasers are the most studied, and they
have demonstrated positive results for nevus of Ota [8]. Lasers are more effective in light-skinned individuals;
however, recurrence can be more common and may re-
sult in a darker hue. 1Our Lady’s Children’s Hospital Crumlin, Dublin, Ireland. 2Oral Medicine
Department, Liverpool University Dental Hospital, Liverpool, UK. 1Our Lady’s Children’s Hospital Crumlin, Dublin, Ireland. 2Oral Medicine
Department, Liverpool University Dental Hospital, Liverpool, UK. Authors’ contributions Nevus of Ota represents a unilateral dermal melanosis in
the distribution of the trigeminal nerve, where it is usu-
ally confined to the ophthalmic and maxillary divisions. The forehead, temple, periorbital area, cheek, and nose
are most commonly involved. Melanin pigmentation in-
volves the eye in about 50% of cases [4]. Rarely is the
pigmentation bilateral with large areas of the face and
oral mucous membranes being affected [2]. JM was involved in assessing the patient, taking photographs, gaining
consent, reviewing the literature, and writing the manuscript. DH was
the consultant in charge and reviewed the literature. Both authors read
and approved the final manuscript. Acknowledgements Acknowledgements
The authors acknowledge Maria Olawale for taking a number of photographs. Acknowledgements Funding
Not applicable. Funding Not applicable. Page 2 of 3 Maguire and Holt Journal of Medical Case Reports (2019) 13:174 Maguire and Holt Journal of Medical Case Reports (2019) 13:174 Maguire and Holt Journal of Medical Case Reports (2019) 13:174 An incisional biopsy of the left buccal mucosa was
completed. The report stated that histological and im-
munohistochemical features were in keeping with a blue
nevus, but within the context of the preexisting occulo-
dermal pigmentation, a diagnosis of oculodermal mela-
nocytosis, also known as “nevus of Ota,” was made. No
other investigations were required. The patient will be
kept under 6-monthly review with the oral medicine
department because the progression of the lesion on the
left buccal mucosa requires active monitoring owing to
the potential for malignant change. to occur with a disproportionate frequency [11]. It is
recommended that these patients be reviewed annu-
ally by a dermatologist. It is also of note that there
are possible molecular explanations for the risk of
malignancy, including mutations of GNAQ and BAP1
genes [12]. This will require further research. 10.
Khwaly JA, Imami N, Shields MB. Glaucoma associated with nevus of Ota.
Arch Ophthalmol. 1995;113(9):1208–9.
11.
Patel CK, et al. Cutaneous malignant melanoma and ocular melanocytosis
(nevus of Ota): report of a case and review of the literature. J Am Acad
Dermatol. 1998;38(5):862–5.
12.
Tse JY, et al. Melanoma arising in a nevus of Ito: novel genetic
mutations and a review of the literature on cutaneous malignant
transformation of dermal melanocytosis. J. Cutan Pathol. 2016;43(1):57–
63. https://doi.org/10.1111/cup.12568. Maguire and Holt Journal of Medical Case Reports (2019) 13:174 References
k 1. Park JH, Lee MH. Acquired, bilateral nevus of Ota-like macules (ABNOM)
associated with Ota’s nevus: case report. Korean Med Sci. 2004;19(4):616–8. 1. Park JH, Lee MH. Acquired, bilateral nevus of Ota-like macules (ABNOM)
associated with Ota’s nevus: case report. Korean Med Sci. 2004;19(4):616–8. 2. Gleason CA, Devaskar SU. Initial evaluation: history and physical examination
of the newborn. In: Avery’s diseases of the newborn. 9th ed. Philadelphia:
Elsevier Saunders; 2012. p. 277–99. Our patient had received laser therapy 20 years earlier
and had a good outcome. She has also been referred to
the cosmetic camouflage clinic. The patient has seen a
makeup artist and uses cosmetics to cover the pig-
mentation. Long-term
follow-up,
especially
in
the
ophthalmology division (6-monthly) [9] and neurosur-
gery division, is required because of the risk of ocular
melanoma and central nervous system neoplasia, al-
though this is rare. The patient’s intracranial lesion is
unrelated to the nevus of Ota. There is also an in-
creased risk of glaucoma associated with nevus of Ota
in 10% of patients [10]. A number of cases of malig-
nant melanoma are also reported in the literature,
and careful observation is necessary, particularly in
Caucasians, in whom malignant degeneration seems 3. Bohra A, Bhateja S. Nevus of Ota’: a rare oro-facial pigmentation –
short review. J Pigment Disord. 2015;2:199. https://doi.org/10.4172/
2376-0427.1000199. 4. Mauropoulos JC, Cohen BA. Disorders of pigmentation. In: Cohen BA, editor. Pediatric dermatology. 4th ed. Philadelphia: Elsevier Saunders. p. 148. 5. Shetty SR, et al. Nevus of Ota with buccal mucosal pigmentation: a rare
case. Dent Res J (Isfahan). 2011;8(1):52–5. 4. Mauropoulos JC, Cohen BA. Disorders of pigmentation. In: Cohen BA, editor. Pediatric dermatology. 4th ed. Philadelphia: Elsevier Saunders. p. 148. gy
p
p
5. Shetty SR, et al. Nevus of Ota with buccal mucosal pigmentation: a rare
case. Dent Res J (Isfahan). 2011;8(1):52–5. 6. Mohan RP, et al. ‘Nevi of Ota: the unusual birthmarks’: a case review. BMJ
Case Rep. 2013;2013:bcr2013008648. 7. Gonder JR, et al. Ocular melanocytosis: a study to determine the prevalence
of ocular melanocytosis. Ophthalmology. 1982;89(8):950–2. 8. Shah VV, Bray FN, Aldahan AS, et al. Lasers and nevus of O
comprehensive review. Lasers Med Sci. 2016;31:179–85. 9. Shields CL, Kaliki S, Livesey M, Walker B, Garoon R, Bucci M, et al. Association of ocular and oculodermal melanocytosis with the rate of
uveal melanoma metastasis: analysis of 7872 consecutive eyes. JAMA
Ophthalmol. 2013;131:993–1003. 10.
Khwaly JA, Imami N, Shields MB. Glaucoma associated with nevus of Ota.
Arch Ophthalmol. 1995;113(9):1208–9. References
k Page 3 of 3 Page 3 of 3 Maguire and Holt Journal of Medical Case Reports (2019) 13:174
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Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells
|
Journal of translational medicine
| 2,011
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cc-by
| 12,387
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Curcumin reduces expression of Bcl-2, leading to
apoptosis in daunorubicin-insensitive CD34+
acute myeloid leukemia cell lines and primary
sorted CD34+ acute myeloid leukemia cells Curcumin reduces expression of Bcl-2, leading to
apoptosis in daunorubicin-insensitive CD34+
acute myeloid leukemia cell lines and primary
sorted CD34+ acute myeloid leukemia cells Jia Rao1,2, Duo-Rong Xu2,3*, Fei-Meng Zheng4, Zi-Jie Long1,2, Sheng-Shan Huang5, Xing Wu1,2, Wei-Hua Zhou1,2,
Ren-Wei Huang1,2* and Quentin Liu1,2,4* Rao et al. Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 * Correspondence: xudr@hotmail.com; huangrw56@163.com; liuq9@mail.sysu.
edu.cn
1Department of Hematology, Third Affiliated Hospital, Sun Yat-sen University,
600 Tianhe Road, Guangzhou 510630, P.R. China
2Sun Yat-sen Institute of Hematology, 600 Tianhe Road, Guangzhou 510630,
P.R. China
Full list of author information is available at the end of the article RESEARCH Open Access © 2011 Rao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Materials Curcumin (Sigma, St. Louis, MO) was dissolved in
dimethyl sulfoxide (DMSO) to prepare a 100-mM stock
solution that was stored at -20°C. DNR was purchased
from Pharmacia & Upjohn SpA (Milan, Italy). Annexin-V
assay kit was purchased from Molecular Probes (Eugene,
OR, USA). Anti-cleaved PARP, cleaved caspase-3, and
Bcl-2 antibodies were purchased from Cell Signaling
Technologies (Beverly, MA, USA). Anti-GAPDH anti-
body and goat anti-rabbit/mouse-horseradish peroxidase
(HRP)-conjugated secondary antibody were purchased
from Protein Tech Group (Chicago, IL, USA). JC-1 kit
was purchased from Beyotime (China). CD34-PE and
IgG1-PE monoclonal antibodies were purchased from BD
Biosciences (San Jose, CA, USA). CD34 MicroBead kit
was purchased from Miltenyi biotec (Auburn, CA, USA). p
CD34+ AML cells are 10-15-fold more resistant to
DNR than CD34- AML cells [8]. CD34+ KG1a and TF-1
AML cell lines are 30-40 fold more resistant to mitox-
antrone than more mature HL-60 and U937 cells, and
this resistance appears to be associated with the lack of
apoptosis [9]. Increasing evidence indicates that CD34+
AML cells are less sensitive to spontaneous apoptosis
and have higher levels of Bcl-2 and Bcl-xl gene and pro-
tein expression than the CD34- subpopulation [6,10-12]. CD34 positivity has been reported to be another indica-
tor of poor prognosis in AML [3,12], and use of more
effective drugs to eliminate this early immature CD34+
AML cell subpopulation might therefore improve the
outcome of AML. Cell lines, primary samples, and cell culture
KG1a and Kasumi-1 cell lines were obtained from
Deutsche Sammlung von Mikroorganismen und Zellkul-
turen GmbH (DSMZ) (Braunschweig, Germany) and
grown in RPMI 1640 medium (Gibco; Invitrogen, Carls-
bad, CA, USA) supplemented with 20% (v/v) fetal bovine
serum (FBS; Hyclone, Logan, UT). According to immu-
nological studies by DSMZ and others [30,31], KG1a and
Kasumi-1 cells are characterized by high expression of
CD34 surface antigen. U937 cells were obtained from the
American Type Culture Collection (ATCC) and grown in
RPMI 1640 medium supplemented with 10% FBS. Cells
were cultured at 37°C in a humidified atmosphere con-
taining 5% CO2. Control cultures received an equivalent
amount of DMSO only. Bone marrow mononuclear cells
(BMMCs) or mobilized peripheral blood mononuclear
cells (PBMCs) were obtained from 9 newly diagnosed
AML patients and 8 healthy donors. All donors provided
written informed consent, and the study had the approval
of the Institute Research Ethics Committee at Sun Yan-
sen University, in accordance with the Declaration of
Helsinki. Abstract Background: Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which
CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin
(DNR) than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in
multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The
aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines
(KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods: Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow
mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT
and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was
analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial
membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related
proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering
RNA (siRNA) against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results: Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a,
Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of
both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells,
consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a
and Kasumi-1 cells to DNR-induced apoptosis. More importantly, curcumin suppressed Bcl-2 expression, selectively
inhibited proliferation and synergistically enhanced the cytotoxicity of DNR in primary CD34+ AML cells, while
showing limited lethality in normal CD34+ hematopoietic progenitors. Conclusion: Curcumin down-regulates Bcl-2 and induces apoptosis in DNR-insensitive CD34+ AML cell lines and
primary CD34+ AML cells. Full list of author information is available at the end of the article © 2011 Rao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Rao et al. Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 2 of 15 Page 2 of 15 Background been found to be a powerful chemosensitizing agent in
tumor cells. It demonstrated no major toxicities in
phase I and II clinical studies at doses of up to 8 g/day
[28,29]. However, the cytotoxic effects of curcumin in
DNR-insensitive CD34+ immature AML cells remain
unclear. Acute myeloid leukemia (AML) is an immunophenotypi-
cally heterogenous malignant disease, in which CD34 posi-
tivity has been significantly correlated with a lower
complete response (CR) rate, drug resistance and poor
outcome [1-3]. Treatment of AML has generally consisted
of a combination of cytarabine and an anthracycline such
as daunorubicin (DNR), or the anthracenedione mitoxan-
trone [4]. Although conventional chemotherapy regimens
induce CR in 65-80% of newly diagnosed AML patients,
most patients who achieve a CR relapse within 2 years
from diagnosis [5]. At relapse, blast cells usually display a
more immature phenotype, with one of the most common
antigenic changes being a gain in expression of the stem
cell antigen CD34 [6,7]. This is reflected in the resistance
of these immature phenotype CD34+ AML progenitors to
current chemotherapies. In this study, we examined the cytotoxic efficiency and
molecular mechanisms underlying the anticancer activity
of curcumin in both DNR-insensitive CD34+ immature
AML cell lines and in primary CD34+AML cells. Wright-Giemsa staining Morphological signs of apoptosis were detected by
Wright-Giemsa staining. Cells were treated with 0-80 μM
curcumin for 24 h. Smears of control and treated cells
were stained with Wright-Giemsa solution for 25 min,
rinsed with distilled water and air dried. Cell morphology
was studied by light microscopy. Detection of mitochondrial membrane potential (MMP,
Δψm) using JC-1 MMP was estimated by flow cytometry after staining
with JC-1 fluorescent dye. When the cell is in a normal
state, MMP is high and JC-1 predominantly appears as
red fluorescence. When the cell is in an apoptotic or
necrotic state, the MMP is reduced and JC-1 appears as a
monomer indicated by green fluorescence. A change in
the florescence from red to green indicates a decrease in
the MMP. Approximately 5×105/ml cells in 6-well plates
were treated with various concentrations of curcumin for
24 h. The cells were then washed with PBS and incubated
with JC-1 working solution for 20 min at 37°C in the
dark. Cells were washed with PBS and resuspended in
500 μl PBS. The stained cells were analyzed by flow cyto-
metry to determine the change in the florescence from
red to green. Colony-forming assay Treated and untreated cells were cultured in RPMI 1640
medium supplemented with 0.9% methylcellulose and
20% FBS at 37°C in 5% CO2. The colonies (containing
50 or more cells) were counted by light microscopy
after 14 days. All semi-solid cultures were performed in
triplicate. Three
independent
experiments
were
performed. Cell cycle analysis Cell cycle was analyzed by flow cytometry. Approxi-
mately 5 × 105/ml cells in 6-well plates were treated
with various concentrations of curcumin for 24 h. Cell
cycle analysis was performed using the CycleTEST™
PLUS DNA kit (BD Biosciences). MTT assay An Annexin V-binding assay was used according to the
manufacturer’s instructions. Briefly, approximately
5×105/ml cells in 6-well plates were treated with various
concentrations of the indicated test samples. The cells
were harvested and used for Annexin V-Alexa Fluor-
488/PI staining. The stained cells were analyzed by flow
cytometry to determine the percentages of AnnexinV
+/PI- (early apoptosis) and AnnexinV+/PI+ (late apopto-
sis) cells. Viability was assessed by MTT assay. Briefly, 1.0×104
cells were incubated in triplicate in a 96-well plate in the
presence or absence of the indicated test samples in a
final volume of 0.2 ml for various lengths of time at 37°C. Thereafter, 20 μl MTT solution (5 mg/ml in PBS) was
then added to each well. After 4-h incubation at 37°C,
150 μl DMSO was added. Finally the plates were shaken
and the optical density at 490 nm was measured using a
multiwell plate reader (Microplate Reader; Bio-Rad,
Hercules, CA). Percent cell viability was calculated as cell
viability of the experimental samples/cell viability of the
control samples × 100. At least three independent experi-
ments were performed. Materials Patient characteristics are shown in Table 1. PBMCs and BMMCs were enriched by Ficoll-Hypaque
density gradient centrifugation and isolated using a CD34 DNR is one of the most commonly used anti-leukemia
agents. Bcl-2 overexpression can block DNR-induced
apoptosis in more mature U937 AML cells [13]. The
anti-apoptotic proteins Bcl-2 and Bcl-xl also contribute
to the survival and chemoresistance of quiescent leuke-
mia CD34+ cells [14]. These findings suggest that Bcl-2
plays a critical role in CD34+ AML cell survival and that
agents aimed at down-regulating Bcl-2 protein might be
effective for the treatment of DNR-insensitive CD34+
AML. Curcumin, a major yellow pigment in turmeric, has
been proven to be a powerful therapeutic drug [15,16]. Curcumin induces apoptosis in a variety of tumor cells,
including more mature HL-60 and U937 cell lines,
through activation of caspase-3, cytochrome c release,
and down-regulation of Bcl-2 [17-20]. Curcumin inhibits
proliferation in a variety of cancer cells through target-
ing multiple cellular signaling pathways [21], including
the mitogen-activated protein kinase [22], nuclear factor
kappaB [23], phosphoinositide-3 kinase/Akt/mammalian
target of rapamycin [24,25], Wnt [26], and Notch-
mediated signaling pathways [27]. Curcumin has also Rao et al. Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 3 of 15 Table 1 Characteristic of patients
Patient#
Age/Sex
FAB
WBC(*109/L)
%CD34 in BMC*
Source
Cytogenetics
P1
35 Y/M
M2
9.09
67.3
BM
46 XY
P2
60 Y/M
M2b
15.80
72.1
BM
46 XY, t(8;21),AML1/ETO #
P3
46 Y/M
M5
12.00
56.0
BM
46 XY
P4
17 Y/M
M5b
3.39
89.1
BM
46 XY
P5
28 Y/M
M2b
11.37
76.3
BM
46 XY, t(8;21),AML1/ETO #
P6
20 Y/M
M1
10.03
70.1
BM
46 XY
P7
78 Y/M
M4
101.08
52.1
PB
46 XY inv (16)(p13q22)
P8
72 Y/F
M2a
1.95
31.5
PB
46 XX
P9
54 Y/M
M1
103.79
62.8
BM
46 XY
P patient, Y year, M male, FAB French-American-British, WBC white blood cells, BMC bone marrow cells, BM bone marrow, PM peripheral blood.*Percentage of
CD34+ cells in bone marrow cells of AML patients before sorting. # The t (8; 21) (q22; q22) chromosomal translocation gives rise to the AML1/ETO fusion
oncoprotein. Table 1 Characteristic of patients P patient, Y year, M male, FAB French-American-British, WBC white blood cells, BMC bone marrow cells, BM bone marrow, PM peripheral blood.*Percentage of
CD34+ cells in bone marrow cells of AML patients before sorting. Materials # The t (8; 21) (q22; q22) chromosomal translocation gives rise to the AML1/ETO fusion
oncoprotein. MicroBead kit. BMMCs and PBMCs were stained with
PE-conjugated anti-CD34 to determine the purity of
CD34+ cells. were washed and stained with Hoechst 33342 (10 μg/ml)
for 15 min at 37°C. Slides were viewed using a fluores-
cence microscope. Western blot analysis Total cellular proteins were isolated with lysis buffer
(20 mM Tris, pH 7.5; 150 mM NaCl; 0.25% NP40;
2.5 mM sodium pyprophosphate; 1 mM EGTA, 1 mM
EDTA; 1 mM b-glycerophosphate; 1 mM Na3VO4; 1 mM
PMSF; 1 μg/ml leupeptin). Equal amounts of protein were
subjected to 10% or 15% sodium dodecyl sulfate-polyacry-
lamide gel electrophoresis and transferred to nitrocellulose
membranes. The membranes were treated with primary
antibodies overnight at 4°C and incubated with a HRP-
conjugated anti-mouse or anti-rabbit secondary antibody
at room temperature for 1 h. The protein bands were
visualized using an enhanced chemiluminescence reagent
(Pierce Biotechnology, USA), according to the manufac-
turer’s instructions. Hoechst 33342 staining Nuclear fragmentation was examined by Hoechst 33342
(Sigma). Cells treated with 0-80 μM curcumin for 24 h Page 4 of 15 Rao et al. Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Results CD34+ KG1a and Kasumi-1cells were insensitive to DNR
KG1a, Kasumi-1 and U937 AML cells were stained with
PE-conjugated CD34 antibody and subjected to flow
cytometry to determine the purity of CD34+ cells. The
percentages of CD34+ cells were 99.43 ± 0.60% in KG1a
cells, 96.67 ± 0.11% in Kasumi-1 cells, but CD34+ was
absent in U937 cells (Figure 1A). After treatment of
these three cell lines with different concentrations of
DNR for 48 h, MTT and apoptosis analyses showed that
DNR inhibited proliferation and induced apoptosis in
more mature U937 cells, but not in immature CD34+
KG1a or Kasumi-1 cell lines (Figure 1B, C). This was in
accord with previous studies indicating that CD34+
AML cells were insensitive to DNR. The concentration
of DNR used in this study was clinically achievable in
patients [37,38]. Statistical analysis RNA isolation and semiquantitative reverse transcription-
polymerase chain reaction (RT-PCR) Data were presented as mean ± SD. One-way ANOVA fol-
lowed by Bonferroni multiple comparison was performed
to assess the differences between two groups under multi-
ple conditions. If the data failed the normality test, the
Kruskal-Wallis one-way ANOVA on ranks was used. A
value of p < 0.05 was considered statistically significant. Both Calcusyn software (Biosoft, Ferguson, MO, USA)
[34,35] and Jin’s formula [36] were used to evaluate the
synergistic effects of drug combinations. Jin’s formula is
given as: Q = Ea + b/(Ea + Eb-Ea × Eb). Ea+b represents
the cell proliferation inhibition rate of the combined
drugs, while Ea and Eb represent the rates for each drug
respectively. A value of Q = 0.85-1.15 indicates a simple
additive effect, while Q > 1.15 indicates synergism. Combi-
nation index (CI) plots were generated using CalcuSyn
software. A value of CI < 1 indicates synergism. Total RNA was extracted using Trizol isolation reagent
(Invitrogen, USA). Reverse transcription was performed
using a reverse transcriptase first strand cDNA synthesis
kit (Takara, Japan). The sequences of the sense and anti-
sense primers were: 5’-CTGGTGGACAACATCGC-3’
(sense) and 5’-GGAGAAATCAAACAGAGGC-3’ (anti-
sense) for Bcl-2, 5’-TGACTTTTCCTGTGAACTCT-3’
(sense) and 5’-GCCTTTCATTCGTATCAAGA-3’ (anti-
sense) for c-IAP-1, 5’-GCAGGGTTTCT TTATACTG-3’
(sense) and 5’-TGTCCCTTCTGTTCTAACAG-3’ (anti-
sense) for XIAP [32], 5’-GTGGACATCCGCAAAGAC-
3’ (sense) and 5’-GAAAGGGTGTAA CGCAACT-3’
(anti-sense) for b-actin. The PCR conditions were as fol-
lows: for c-IAP-1 and XIAP, 94°C for 1 min, 62°C for
1 min, and 72°C for 1 min; for Bcl-2, 94°C for 30 s,
62°C for 30 s, 72°C for 10 s; and for b-actin: 94°C for
30 s, 55°C for 30 s, 72°C for 1 min. Thirty cycles of
amplification were used. PCR (10 μl) products were ana-
lyzed by electrophoresis on 2% (w/v) agarose gel. Short interfering RNA (siRNA) transfection KG1a and Kasumi-1 cells were seeded onto 6-well plates
for 24 h before transfection. Control scrambled siRNA
was synthesized and purchased from GenePharma (Shang-
hai Co. Ltd., China). SiRNA Bcl-2 (50 nM): 5’-GGGA-
GAUAGUGAUGAAG UAUU-3’ [33] or control scramble
sequences were transfected using Lipofectamine 2000
reagent (Invitrogen), according to the manufacturer’s pro-
tocol. Briefly, for each well, 5 μl Lipofectamine 2000 was
diluted in 250 μl Opti-MEM medium (Invitrogen). The
mixture was gently added to a solution containing siRNA
in 250 μl Opti-MEMI medium and incubated for 20 min. The mixture was then added to the plates. After transfec-
tion with siRNA for 24 h, cells were harvested for further
assay. KG1a, Kasumi-1 and U937 cell lines were exposed to
curcumin (0-100 μM) for 24, 48, 72 and 96 h. The cyto-
toxic effects of curcumin were determined by MTT
assay. Curcumin had a significant cytotoxic effect in all
tested cell lines in both dose- and time-dependent man-
ners (Figure 2B, C, D). The IC50 values at 24, 48, 72,
and 96 h were 230.5, 86.9, 60.0, and 35.7 μM for KG1a,
68.5, 46.6, 28.8, and 23.5 μM for Kasumi-1, and 58.3,
26.0, 10.6, and 4.4 μM for U937 cells, respectively. The
antiproliferative effects of curcumin in these cell lines
were further determined using clonogenic assays. Curcu-
min inhibited clonogenic growth in a dose-dependent
manner, and completely inhibited colony formation at a
dose as low as 20 μM (Figure S1A, B, Additional file 1). Rao et al. Short interfering RNA (siRNA) transfection Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 5 of 15 Cell cycle distributions in KG1a, Kasumi-1, and U937
cells were examined after treatment with curcumin for
and a decrease in the percentage of cells in the S phase,
from 39-23% Similar results were found for Kasumi-1
M1
A
B
M1
M1
KG1a
U937
Kasumi-1
99.72
0.18
.*D
NDVXPL
X
Cell viability(%;MTT
assay)
***
***
***
Kasumi-1
Apoptosis(%)
.*D
NDVXPL
8
***
***
***
96.71
Daunorubicin(ȝg/ml)
Daunorubicin(ȝg/ml)
KG1a
U937
C
Annexin č
PI
0 0.4 0.8 1.6
Daunorubicin(ȝg/ml)
1.65
8.13
0.02
90.19
1.35
10.15
0.00
88.50
4.84
0.89
94.27
0.00
0.37
2.69
26.94
0.00
1.32
4.65
0.01
94.02
0.69
3.64
0.00
95.67
0.42
3.51
0.00
96.07
0.56
3.69
0.05
95.70
14.16
26.56
12.68
46.60
12.39
16.13
48.40
23.08
8.78
19.60
1.62
69.99
0.64
2.75
0.01
96.60
Figure 1 CD34+ KG1a and Kasumi-1cells were insensitive to DNR. (A) KG1a, Kasumi-1 and U937 cells were stained with PE-conjugated CD34
antibody and subjected to flow cytometry to determine the purity of CD34+ cells. (B, C) These three cell lines were treated with different
concentrations of DNR for 48 h. MTT assay (B) was performed as described in “Methods” and apoptosis (C) was assessed by Annexin V/PI assays. Cells in the lower right quadrant represent early apoptosis and those in the upper right quadrant represent late apoptosis. The graph displays
the means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (compared with control). Short interfering RNA (siRNA) transfection Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 6 of 15
K
K
K
K
*0
6
**
Curcumin(ȝM)
K
K
K
K
C
Cell viability(%;MTT
assay)
Cell viability(%;MTT
assay)
KG1a
Kasumi-1
A
Curcumin
Curcumin(ȝM)
E
KG1a Kasumi-1 U937
Curcumin(ȝM)
D
K
K
K
K
B
Cell viability(%;MTT
assay)
Curcumin(ȝM)
U937
Figure 2 Curcumin suppressed cell growth and induced G1/S arrest. (A) Structure of curcumin. (B, C, D) KG1a, Kasumi-1 and U937 cell lines
were treated with different concentrations of curcumin for 24, 48, 72, and 96 h. MTT assay was performed. (E) These three cell lines were treated
with different concentrations of curcumin for 24 h and analyzed for DNA content by flow cytometry, as described in “Methods.” The bar
represents means ± SD of three independent experiments. Rao et al. Short interfering RNA (siRNA) transfection M1
A
M1
M1
KG1a
U937
Kasumi-1
99.72
0.18
96.71 A B
.*D
NDVXPL
X
Cell viability(%;MTT
assay)
***
***
***
Daunorubicin(ȝg/ml) B Kasumi-1
Apoptosis(%)
.*D
NDVXPL
8
***
***
***
Daunorubicin(ȝg/ml)
KG1a
U937
C
Annexin č
PI
0 0.4 0.8 1.6
Daunorubicin(ȝg/ml)
1.65
8.13
0.02
90.19
1.35
10.15
0.00
88.50
4.84
0.89
94.27
0.00
0.37
2.69
26.94
0.00
1.32
4.65
0.01
94.02
0.69
3.64
0.00
95.67
0.42
3.51
0.00
96.07
0.56
3.69
0.05
95.70
14.16
26.56
12.68
46.60
12.39
16.13
48.40
23.08
8.78
19.60
1.62
69.99
0.64
2.75
0.01
96.60 C Annexin č Annexin č Figure 1 CD34+ KG1a and Kasumi-1cells were insensitive to DNR. (A) KG1a, Kasumi-1 and U937 cells were stained with PE-conjugated CD34
antibody and subjected to flow cytometry to determine the purity of CD34+ cells. (B, C) These three cell lines were treated with different
concentrations of DNR for 48 h. MTT assay (B) was performed as described in “Methods” and apoptosis (C) was assessed by Annexin V/PI assays. Cells in the lower right quadrant represent early apoptosis and those in the upper right quadrant represent late apoptosis. The graph displays
the means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (compared with control). Cell cycle distributions in KG1a, Kasumi-1, and U937
cells were examined after treatment with curcumin for
24 h. As shown in Figure 2E, treatment of KG1a cells
with 80 μM curcumin resulted in a significant increase
in the percentage of cells in the G1 phase, from 46-62%, Cell cycle distributions in KG1a, Kasumi-1, and U937
cells were examined after treatment with curcumin for
24 h. As shown in Figure 2E, treatment of KG1a cells
with 80 μM curcumin resulted in a significant increase
in the percentage of cells in the G1 phase, from 46-62%, and a decrease in the percentage of cells in the S phase,
from 39-23%. Similar results were found for Kasumi-1
and U937 cells. These results demonstrated that curcu-
min induced G1/S arrest in both DNR-insensitive and
-sensitive AML cell lines. Rao et al. Short interfering RNA (siRNA) transfection Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Curcumin induced apoptosis through activation of
caspase-3 followed by PARP degradation in both DNR-
insensitive and -sensitive AML cell lines cells were cultured with combinations of these two
drugs at different doses but in a constant ratio (curcu-
min to DNR: 20 μM to 0.1 μg/ml, 40 μM to 0.2 μg/ml,
and 80 μM to 0.4 μg/ml, respectively) for 48 h, as
shown in Figure 5A, B and Table S1 (Additional file 3). Both CalcuSyn software and Jin’s formula were used to
determine synergy, and the results were consistent. With the exception of co-treatment of KG1a cells with
20 μM curcumin and 0.1 μg/ml DNR, which showed an
additive effect (CI = 1.03, Q = 0.99), co-treatment with
other doses in KG1a cells and with all doses in Kasumi-
1 cells exhibited synergistic effects. For example, the
combination of 40 μM curcumin with 0.2 μg/ml DNR
in KG1a cells caused growth inhibition of 45.12%, com-
pared to curcumin (26.31%) or DNR (5.47%) alone, indi-
cating synergism (CI = 0.654, Q = 1.49). Notably, co-
treatment with 40 μM curcumin and 0.2 μg/ml DNR
caused more attenuation of Bcl-2 protein levels than
treatment with either agent alone (Figure 5C). To determine if growth inhibition induced by curcumin
was a result of apoptosis, the pro-apoptotic effect was
examined using Wright-Giemsa, Hoechst 33342 and
Annexin-V/PI staining. Both Wright-Giemsa and
Hoechst 33342 staining showed that curcumin induced
morphological changes such as cell shrinkage and nuclear
condensation, which are typical characteristics of apopto-
sis (Figure 3A; Figure S2A, Additional file 2). These mor-
phological changes were confirmed by flow cytometry. Treatment with curcumin at 40 μM for 24 h resulted in
apoptosis rates of 23.5 ± 8.8%, 36.1 ± 5.3%, and 40.1 ±
17.8% in KG1a, Kasumi-1 and U937 cells, respectively
(Figure 3B). Western blotting analysis further showed
that curcumin induced caspase-3 activation and PARP
cleavage, two hallmarks of apoptosis (Figure 3C). Both
Annexin-V/PI and Western blotting showed that curcu-
min induced apoptosis in a dose-dependent manner. U937cells were the most sensitive to curcumin-induced
apoptosis, followed by Kasumi-1, then KG1a cells. Suppression of Bcl-2 with siRNA induced apoptosis and
increased the susceptibility of KG1a and Kasumi-1 cells to
DNR-induced apoptosis Short interfering RNA (siRNA) transfection Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71
Page 6 of 15 A
Curcumin
B
K
K
K
K
B
Cell viability(%;MTT
assay)
Curcumin(ȝM)
U937 A
Curcumin
K
K
K
K
B
Cell viability(%;MTT
assay)
Curcumin(ȝM)
U937 A B Curcumin(ȝM)
K
K
K
K
Curcumin(ȝM)
K
K
K
K
C
Cell viability(%;MTT
assay)
Cell viability(%;MTT
assay)
KG1a
Kasumi-1
Curcumin(ȝM)
D
K
K
K
K
Curcumin(ȝM)
Cell viability(%;MTT
assay)
KG1a
D
K
K
K
K
C
Cell viability(%;MTT
assay)
Kasumi-1
Curcumin(ȝM)
D D C Curcumin(ȝM)
*0
6
**
E
KG1a Kasumi-1 U937
Curcumin(ȝM) E Figure 2 Curcumin suppressed cell growth and induced G1/S arrest. (A) Structure of curcumin. (B, C, D) KG1a, Kasumi-1 and U937 cell lines
were treated with different concentrations of curcumin for 24, 48, 72, and 96 h. MTT assay was performed. (E) These three cell lines were treated
with different concentrations of curcumin for 24 h and analyzed for DNA content by flow cytometry, as described in “Methods.” The bar
represents means ± SD of three independent experiments. Figure 2 Curcumin suppressed cell growth and induced G1/S arrest. (A) Structure of curcumin. (B, C, D) KG1a, Kasumi-1 and U937 cell lines
were treated with different concentrations of curcumin for 24, 48, 72, and 96 h. MTT assay was performed. (E) These three cell lines were treated
with different concentrations of curcumin for 24 h and analyzed for DNA content by flow cytometry, as described in “Methods.” The bar
represents means ± SD of three independent experiments. Page 7 of 15 Rao et al. Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Rao et al. Suppression of Bcl-2 with siRNA induced apoptosis and
increased the susceptibility of KG1a and Kasumi-1 cells to
DNR-induced apoptosis To clarify if down-regulation of Bcl-2 by curcumin plays
an important role in this synergistic effect, Bcl-2 expres-
sion was suppressed by siRNA and the effect on apopto-
sis and DNR sensitivity was examined by flow cytometry. Bcl-2 siRNA-induced apoptosis in 24 h (28.58% in KG1a
cells, 37.12% in Kasumi-1 cells) was similar to that in cur-
cumin-treated KG1a (31.71%, 60 μM, Figure 3B) and
Kasumi-1 cells (36.10%, 40 μM, Figure 3B), respectively
(Figure 6A, B). As shown in Figure 6C, suppression of
Bcl-2 by siRNA increased the susceptibility of these cell
lines to DNR-induced apoptosis (40.15% in KG1a cells
and 86.23% in Kasumi-1 cells), compared to DNR only
(3.17% in KG1a cells, 5.94% in Kasumi-1 cells). These
results suggest that suppression of Bcl-2 could contribute
to curcumin-induced apoptosis and the synergistic effect
of curcumin and DNR. Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines The mechanisms underlying curcumin-induced apopto-
sis were investigated. The IAP and Bcl-2 family play an
important role in the regulation of cell apoptosis, and
the effects of curcumin on mRNA levels of c-IAP-1,
XIAP and Bcl-2 were therefore assessed by RT-PCR. As
shown in Figure 4A, Bcl-2 mRNA levels were signifi-
cantly down-regulated in both DNR-insensitive AML
cell lines (KG1a and Kasumi-1) and in DNR-sensitive
U937 cells, while the levels of c-IAP-1 and XIAP were
unchanged. Western blotting also demonstrated that
curcumin significantly down-regulated Bcl-2 protein
levels in a dose-dependent manner (Figure 4B). These
results suggest that down-regulation of Bcl-2 could con-
tribute to curcumin-induced apoptosis. Curcumin was effective against primary CD34+ AML cells
The cytotoxic effects of either curcumin and/or DNR on
primary CD34+AML cells were also examined. CD34+
cells were sorted from BMMCs or PBMCs from 9 AML
patients and 8 healthy donors. The sorted samples yielded
more than 95% CD34+ cells with greater than 90% viabi-
lity, determined by trypan blue exclusion (Figure 7A). The
antiproliferative effects of curcumin on CD34+ cells from
3 AML patients (patients 1, 2, 3) and 3 healthy donors
(donors 1, 2, 3) were determined by MTT assay, and com-
pared with the results of DNR treatment. CD34+ cells
were treated with curcumin (0, 10, 20, 40, 80 μM) or DNR
(0, 0.4, 0.8, 1.6 μg/ml) for 24 h. Curcumin significantly
inhibited proliferation of CD34+ AML cells, but only Disruption of the function of Bcl-2 protein leads to per-
meabilization of the mitochondrial membrane [39]. We
therefore investigated the effects of curcumin on MMP
using JC-1 fluorescent dye and flow cytometry. Exposure
of the three cell lines to increasing doses of curcumin for
24 h led to a significant reduction in the MMP (Figure
4C). These results suggest that curcumin-induced apopto-
sis is mitochondria-dependent. Curcumin synergistically enhanced the cytotoxic effect of
DNR in DNR-insensitive KG1a and Kasumi-1 cells,
associated with down-regulation of Bcl-2 To determine if curcumin could enhance the cytotoxic
activity of DNR, DNR-insensitive KG1a and Kasumi-1 Rao et al. Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 8 of 15 Kasumi-1
KG1a
0 40 60 80
A
U937
Curcumin(ȝM) Kasumi-1
KG1a
0 40 60 80
A
U937
Curcumin(ȝM) A
.*D
.DVXPL
8
Apoptosis(%)
Kasumi-1
KG1a
0 40 60 80
A
U937
C
Curcumin(ȝM)
Curcumin(ȝM)
Cleaved caspase-3
0 40 60 80 0 40 60 80 0 40 60 80
KG1a Kasumi-1 U937
Cleaved PARP
GAPDH
Curcumin(ȝM)
Annexin č
PI
B
KG1a
U937
0
40
60
80
Kasumi-1
Curcumin(ȝM)
0.26
91.83
2.36
5.55
2.99
19.80
1.01
76.2
25.78
70.89
3.83
38.27
0.56
57.34
2.66
0.67
76.79
12.83
0.04
1.88
93.59
4.49
19.96
0.90
25.08
54.06
82.29
12.26
0.43
9.95
0.15
5.30
1.81
7.09
91.09
0.02
7.93
21.09
0.03
70.95
17.22
31.28
0.03
51.47
23.89
34.56
41.55
0.00
Figure 3 Curcumin induced apoptosis through activation of caspase-3 followed by PARP degradation. KG1a, Kasumi-1 and U937 cells
were incubated with indicated concentrations of curcumin for 24 h. (A) Cells were stained with Wright-Giemsa and then examined under a
light microscope. Arrows indicate apoptotic cells (magnification ×400). (B) Cells were stained with Annexin V/PI to analyze apoptotic cell
populations. The graph displays the means ± SD of four independent experiments. (C) Western blotting analysis showed cleaved caspase-3 (17,
19 kDa) and cleaved PARP (89 kDa) fragment. Three independent experiments were performed with similar results, and representative data are
shown. Kasumi-1 B
.*D
.DVXPL
8
Apoptosis(%)
Curcumin(ȝM)
Annexin č
PI
B
KG1a
U937
0
40
60
80
Kasumi-1
Curcumin(ȝM)
0.26
91.83
2.36
5.55
2.99
19.80
1.01
76.2
25.78
70.89
3.83
38.27
0.56
57.34
2.66
0.67
76.79
12.83
0.04
1.88
93.59
4.49
19.96
0.90
25.08
54.06
82.29
12.26
0.43
9.95
0.15
5.30
1.81
7.09
91.09
0.02
7.93
21.09
0.03
70.95
17.22
31.28
0.03
51.47
23.89
34.56
41.55
0.00 KG1a
Kasumi-1 Kasumi-1
C
Curcumin(ȝM)
Cleaved caspase-3
0 40 60 80 0 40 60 80 0 40 60 80
KG1a Kasumi-1 U937
Cleaved PARP
GAPDH
Curcumin(ȝM)
Annexin č
U9
76.79
93.59
4.49
25.08
54.06
82.29
12.26
9.95
Figure 3 Curcumin induced apoptosis through activation of caspase-3 followed by PARP degradation. KG1a, Kasumi-1 and U937 cells
were incubated with indicated concentrations of curcumin for 24 h. Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines (A) Cells were stained with Wright-Giemsa and then examined under a
light microscope. Arrows indicate apoptotic cells (magnification ×400). (B) Cells were stained with Annexin V/PI to analyze apoptotic cell
populations. The graph displays the means ± SD of four independent experiments. (C) Western blotting analysis showed cleaved caspase-3 (17,
19 kDa) and cleaved PARP (89 kDa) fragment. Three independent experiments were performed with similar results, and representative data are
Curcumin(ȝM) C
Cleaved caspase-3
0 40 60 80 0 40 60 80 0 40 60 80
KG1a Kasumi-1 U937
Cleaved PARP
GAPDH
Curcumin(ȝM) C Figure 3 Curcumin induced apoptosis through activation of caspase-3 followed by PARP degradation. KG1a, Kasumi-1 and U937 cells
were incubated with indicated concentrations of curcumin for 24 h. (A) Cells were stained with Wright-Giemsa and then examined under a
light microscope. Arrows indicate apoptotic cells (magnification ×400). (B) Cells were stained with Annexin V/PI to analyze apoptotic cell
populations. The graph displays the means ± SD of four independent experiments. (C) Western blotting analysis showed cleaved caspase-3 (17,
19 kDa) and cleaved PARP (89 kDa) fragment. Three independent experiments were performed with similar results, and representative data are
shown. Page 9 of 15 Rao et al. Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 ___________________
0 40 60 80
___________________
0 40 60 80
KG1a kasumi-1 U937
.*D
NDVXPL
8
B
A
____________________
0 40 60 80
Bcl-2
c-IAP-1
XIAP
ȕ-actin
Red/green ratio
(% of control)
C
Curcumin(ȝM)
Curcumin(ȝM)
GAPDH
Bcl-2
0 40 60 80 0 40 60 80
KG1a kasumi-1 U937
Curcumin(ȝM)
0 40 60 80
Green
Red
0
40
60
80
KG1a
Kasumi-1
Curcumin(ȝM)
U937
38.27
19.14
80.82
23.86
76.13
37.65
62.33
93.25
6.47
49.91
97.31
50.05
72.29
27.68
92.88
7.08
2.58
0.36
99.63
7.25
92.74
20.25
79.73
84.76
15.22
Figure 4 Curcumin decreased Bcl-2 mRNA and protein levels and caused the loss of MMP. KG1a, Kasumi-1 and U937 cells were exposed
to different concentrations of curcumin for 24 h. (A) The effects on Bcl-2, c-IAP-1, and XIAP mRNA levels were determined by RT-PCR. (B) The
effect on Bcl-2 protein levels was determined by Western blotting assay. Three independent experiments were performed with similar results,
and representative data are shown. (C) MMP was estimated by flow cytometry showing decrease in the red to green fluorescence ratio. Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines The
results shown are representative of three independent experiments. The bar represents mean ± SD of three independent experiments. ___________________
0 40 60 80
___________________
0 40 60 80
KG1a kasumi-1 U937
A
____________________
0 40 60 80
Bcl-2
c-IAP-1
XIAP
ȕ-actin
Curcumin(ȝM) A B B
GAPDH
Bcl-2
0 40 60 80 0 40 60 80
KG1a kasumi-1 U937
Curcumin(ȝM)
0 40 60 80 C
Green
Red
0
40
60
80
KG1a
Kasumi-1
Curcumin(ȝM)
U937
38.27
19.14
80.82
23.86
76.13
37.65
62.33
93.25
6.47
49.91
97.31
50.05
72.29
27.68
92.88
7.08
2.58
0.36
99.63
7.25
92.74
20.25
79.73
84.76
15.22 C KG1a U937
.*D
NDVXPL
8
Red/green ratio
(% of control)
Curcumin(ȝM)
Curcumin(ȝM) Figure 4 Curcumin decreased Bcl-2 mRNA and protein levels and caused the loss of MMP. KG1a, Kasumi-1 and U937 cells were exposed
to different concentrations of curcumin for 24 h. (A) The effects on Bcl-2, c-IAP-1, and XIAP mRNA levels were determined by RT-PCR. (B) The
effect on Bcl-2 protein levels was determined by Western blotting assay. Three independent experiments were performed with similar results,
and representative data are shown. (C) MMP was estimated by flow cytometry showing decrease in the red to green fluorescence ratio. The
results shown are representative of three independent experiments. The bar represents mean ± SD of three independent experiments. Rao et al. Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 10 of 15 A
c
a
b
KG1a Bcl-2
GAPDH
– + – +
– – + +
– + – +
– – + +
KG1a Kasumi-1
Daunorubicin(0.2ȝg/ml)
Curcumin(40ȝM)
C
A
c
a
b
a
b
c
KG1a
Kasumi-1
B
Figure 5 Curcumin synergistically enhanced the cytotoxic effects of DNR associated with down-regulation of Bcl-2. KG1a and Kausumi-
1cells were exposed to different concentrations of curcumin, DNR, or their combination as indicated, for 48 h. (A, B) CI-effect plots and median-
effect plots were generated using CalcuSyn software. The points a, b, and c represent CI values for the combinations 20, 40, and 80 μM
curcumin with 0.1, 0.2, and 0.4 μg/ml DNR in a constant ratio, respectively. The CI values at ED50, ED75, ED90 were 0.667, 0.490, and 0.364 for
KG1a cells and 0.529, 0.456, and 0.394 for Kasumi-1 cells, respectively. (C) Bcl-2 protein levels were determined by Western blotting assay. Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines Three
independent experiments were performed with similar results, and representative data are shown. A B Kasumi-1 C Daunorubicin(0.2ȝg/ml)
Curcumin(40ȝM) Figure 5 Curcumin synergistically enhanced the cytotoxic effects of DNR associated with down-regulation of Bcl-2. KG1a and Kausumi-
1cells were exposed to different concentrations of curcumin, DNR, or their combination as indicated, for 48 h. (A, B) CI-effect plots and median-
effect plots were generated using CalcuSyn software. The points a, b, and c represent CI values for the combinations 20, 40, and 80 μM
curcumin with 0.1, 0.2, and 0.4 μg/ml DNR in a constant ratio, respectively. The CI values at ED50, ED75, ED90 were 0.667, 0.490, and 0.364 for
KG1a cells and 0.529, 0.456, and 0.394 for Kasumi-1 cells, respectively. (C) Bcl-2 protein levels were determined by Western blotting assay. Three
independent experiments were performed with similar results, and representative data are shown. Rao et al. Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 11 of 15
VLFRQWURO
VLFRQWURO
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VL%FO
.*D
.DVXPL
– + – +
Apoptosis(%)
Daunorubicin(0.2ȝg/ml)
C
– + – +
Si-Bcl-2
Si-control
KG1a
Kasumi-1
Daunorubicin(0.2ȝg/ml)
0.29
0.74
86.35
12.62
2.44
45.76
42.06
9.74
1.60
9.19
18.77
70.44
1.13
5.73
11.86
81.28
1.25
97.55
97.90
1.38
6.35
76.31
22.63
12.43
60.82
0.61
0.11
1.11
0.09
6.76
10.58
4.12
A
Si-control Si-Bcl-2
Bcl-2
GAPDH
KG1a
Si-contril Si-Bcl-2
Bcl-2
GAPDH
Kasumi-1
Apoptosis(%)
.*D
.DVXPLˉ
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VL%FO
B
Si-control Si-Bcl-2
KG1a
Kasumi-1
1.44
0.60
0.06
97.9
1.01
0.05
1.85
97.09
0.26
6.86
20.08
72.80
24.29
28.17
1.15
46.39
Figure 6 Suppression of Bcl-2 with siRNA induced apoptosis and increased susceptibility to DNR. KG1a and Kasumi-1cells were
transfected with siRNA Bcl-2 or siRNA control for 24 h. (A) Bcl-2 protein levels were determined by Western blotting assay. (B) Cells were stained
with Annexin V/PI to analyze apoptotic cell populations. The graph displays the means ± SD of three independent experiments. (C) KG1a and
Kasumi-1 cells were transfected with siRNA Bcl-2 or siRNA control for 24 h, and then treated with DNR (0.2 μg/ml) for 48 h. Cells were stained
with Annexin V/PI to analyze apoptotic cell populations. The bar represents mean ± SD of three independent experiments. ** p < 0.01, *** p <
0.001 (compared with control). Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines http://www.translational-medicine.com/content/9/1/71 A
Si-control Si-Bcl-2
Bcl-2
GAPDH
KG1a
Si-contril Si-Bcl-2
Bcl-2
GAPDH
Kasumi-1
Apoptosis(%)
.*D
.DVXPLˉ
VLFRQWURO
VL%FO
B
Si-control Si-Bcl-2
KG1a
Kasumi-1
1.44
0.60
0.06
97.9
1.01
0.05
1.85
97.09
0.26
6.86
20.08
72.80
24.29
28.17
1.15
46.39 A
Si-control Si-Bcl-2
Bcl-2
GAPDH
KG1a
Si-contril Si-Bcl-2
Bcl-2
GAPDH
Kasumi-1 Apoptosis(%)
.*D
.DVXPLˉ
VLFRQWURO
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B
Si-control Si-Bcl-2
KG1a
Kasumi-1
1.44
0.60
0.06
97.9
1.01
0.05
1.85
97.09
0.26
6.86
20.08
72.80
24.29
28.17
1.15
46.39
.*D
C
– + – +
Si-Bcl-2
Si-control
KG1a
Kasumi-1
Daunorubicin(0.2ȝg/ml)
0.29
0.74
86.35
12.62
2.44
45.76
42.06
9.74
1.60
9.19
18.77
70.44
1.13
5.73
11.86
81.28
1.25
97.55
97.90
1.38
6.35
76.31
22.63
12.43
60.82
0.61
0.11
1.11
0.09
6.76
10.58
4.12
A
Si-control Si-Bcl-2
Bcl-2
GAPDH
KG1a
Si-contril Si-Bcl-2
Bcl-2
GAPDH
Kasumi-1
Apoptosis(%)
.*D
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Si-control Si-Bcl-2
KG1a
Kasumi-1
1.44
0.60
0.06
97.9
1.01
0.05
1.85
97.09
0.26
6.86
20.08
72.80
24.29
28.17
1.15
46.39 A B
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transfected with siRNA Bcl-2 or siRNA control for 24 h. (A) Bcl-2 protein levels were determined by Western blotting assay. (B) Cells were stained
with Annexin V/PI to analyze apoptotic cell populations. The graph displays the means ± SD of three independent experiments. (C) KG1a and
Kasumi-1 cells were transfected with siRNA Bcl-2 or siRNA control for 24 h, and then treated with DNR (0.2 μg/ml) for 48 h. Cells were stained
with Annexin V/PI to analyze apoptotic cell populations. The bar represents mean ± SD of three independent experiments. ** p < 0.01, *** p <
0.001 (compared with control). Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines
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transfected with siRNA Bcl-2 or siRNA control for 24 h. (A) Bcl-2 protein levels were determined by Western blotting assay. (B) Cells were stained
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4.12 Daunorubicin(0.2ȝg/ml) Kasumi-1 Apoptosis(%) Daunorubicin(0.2ȝg/ml) Figure 6 Suppression of Bcl-2 with siRNA induced apoptosis and increased susceptibility to DNR. KG1a and Kasumi-1cells were
transfected with siRNA Bcl-2 or siRNA control for 24 h. (A) Bcl-2 protein levels were determined by Western blotting assay. (B) Cells were stained
with Annexin V/PI to analyze apoptotic cell populations. The graph displays the means ± SD of three independent experiments. (C) KG1a and
Kasumi-1 cells were transfected with siRNA Bcl-2 or siRNA control for 24 h, and then treated with DNR (0.2 μg/ml) for 48 h. Cells were stained
with Annexin V/PI to analyze apoptotic cell populations. The bar represents mean ± SD of three independent experiments. ** p < 0.01, *** p <
0.001 (compared with control). Rao et al. Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 12 of 15
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patients and 8 healthy donors were isolated and subjected to flow cytometry to determine the purity of CD34+ cells. (B) 3 CD34+ AML and 3
CD34+ normal samples were treated with different concentrations of curcumin (0, 10, 20, 40, and 80 μM) for 24 h. The same CD34+ AML
samples were also exposed to DNR (0, 0.4, 0.8, and 1.6 μg/ml) for 24 h. MTT assay was performed. The bar represents mean ± SD of three
independent experiments. (C) Another set of 3 CD34+ AML samples and 3 CD34+ normal samples were exposed to different concentrations of
curcumin, DNR, and their combination as indicated, for 48 h. MTT assay was performed. * P < 0.05, ** P < 0.01 (compared with either curcumin
or DNR alone). (D, E) 4 CD34+ AML samples and 3 CD34+ normal samples were treated with 0 and 40 μM curcumin for 24 h. Apoptosis was
assessed by AnnexinV/PI assay. The graph displays the mean ± SD of four independent experiments (D). A representative figure is shown (E). (F)
3 CD34+ AML samples were treated with 0 and 80 μM curcumin for 24 h. Bcl-2 protein levels were determined by Western blotting assay. Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 13 of 15 intrinsic apoptosis pathway involving down-regulation of
Bcl-2 protein, loss of MMP and activation of caspase-3,
followed by PARP degradation. Furthermore, suppres-
sion of Bcl-2 with siRNA caused significant apoptosis,
similar to that seen in curcumin-treated cells, suggesting
an important role for Bcl-2 in curcumin-induced apop-
tosis in these CD34+AML cell lines. exhibited modest lethality in normal CD34+ hematopoietic
progenitors (Figure 7B). However, CD34+ cells derived
from the 3 AML patients were insensitive to DNR (Figure
7B). Synergy between curcumin and DNR was examined
in another set of 3 AML patients (patients 7, 8, 9) and 3
healthy donors (donors 6, 7, 8). CD34+ cells were treated
with curcumin (0, 10, 20, 40, 80 μM) and/or DNR (0.2 μg/
ml) for 48 h. Curcumin at 20, 40, or 80 μM synergistically
enhanced the cytotoxic effect of DNR (0.2 μg/ml) in CD34
+ AML cells, with Q values of 1.60, 1.35 and 1.33, respec-
tively. Normal CD34+ progenitors were less susceptible to
the combined toxic effects (Figure 7C). 4 AML patients
(patients 4, 5, 6, 7) and 3 donors (donors 4, 5, 6) yielded
sufficient numbers of cells for apoptosis assay by flow
cytometry. As shown in Figure 7D, E, curcumin induced
significant apoptosis in CD34+ AML cells, but minimal
apoptosis in normal CD34+ hematopoietic progenitors. 3
AML samples (patients 5, 7, 8) with sufficient cell num-
bers were further analyzed for Bcl-2 protein expression by
Western blotting assay. A dose of 80 μM curcumin was
used in primary CD34+ AML cells, because curcumin sig-
nificantly down-regulated the Bcl-2 protein levels in CD34
+ AML cell lines at 80 μM. The results showed that treat-
ment with 80 μM curcumin significantly down-regulated
Bcl-2 protein levels (Figure 7F). Accumulating evidence has shown that curcumin
potentiates the effects of chemotherapeutic drugs such
as bortezomib, cisplatin, and 5-fluorouracil plus oxali-
platin (FOLFOX) in vitro and vivo [40-42]. Notably, Yu
et al. revealed that curcumin, either alone or together
with FOLFOX, could effectively eliminate FOLFOX-
resistant colon cancer stem cells (CSCs) [42]. CSCs have
been proposed to be responsible for disease progression
or relapse following conventional therapy [43], and the
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patients and 8 healthy donors were isolated and subjected to flow cytometry to determine the purity of CD34+ cells. (B) 3 CD34+ AML and 3
CD34+ normal samples were treated with different concentrations of curcumin (0, 10, 20, 40, and 80 μM) for 24 h. The same CD34+ AML
samples were also exposed to DNR (0, 0.4, 0.8, and 1.6 μg/ml) for 24 h. MTT assay was performed. The bar represents mean ± SD of three
independent experiments. (C) Another set of 3 CD34+ AML samples and 3 CD34+ normal samples were exposed to different concentrations of
curcumin, DNR, and their combination as indicated, for 48 h. MTT assay was performed. * P < 0.05, ** P < 0.01 (compared with either curcumin
or DNR alone). (D, E) 4 CD34+ AML samples and 3 CD34+ normal samples were treated with 0 and 40 μM curcumin for 24 h. Apoptosis was
assessed by AnnexinV/PI assay. The graph displays the mean ± SD of four independent experiments (D). A representative figure is shown (E). (F)
3 CD34+ AML samples were treated with 0 and 80 μM curcumin for 24 h. Bcl-2 protein levels were determined by Western blotting assay. Rao et al. Curcumin decreased Bcl-2 mRNA and protein levels and
reduced MMP in both DNR-insensitive and -sensitive AML
cell lines A recent study indicated
that the combination of curcumin with carnosic acid
also produced a synergistic antiproliferative effect on
KG1a cells; however, this synergism was not associated
with alterations in Bcl-2 levels [44]. In contrast, our
study demonstrated that curcumin synergistically
enhanced the cytotoxic effects of DNR in association
with decreased Bcl-2 expression in KG1a and Kasumi-1
cells. Accordingly, siRNA against Bcl-2 increased the
susceptibility of these CD34+ cell lines to DNR-induced
apoptosis, indicating that Bcl-2 down-regulation played
an important role in this curcumin-induced synergistic
effect. Discussion
CD3
i i CD34 positivity has been reported to be an indicator of
poor prognosis in AML [3]. In the present study, we
evaluated the cytotoxicity of curcumin in DNR-insensi-
tive CD34+ AML cell lines (KG1a and Kasumi-1) and in
CD34+ primary AML samples. We showed that curcu-
min selectively induced apoptosis in KG1a and Kasumi-
1 cell lines, as well as in primary CD34+ AML cells, in
association with down-regulation of Bcl-2 expression. Importantly, co-treatment with curcumin and DNR
synergistically inhibited proliferation, consistent with
decreased Bcl-2 expression. Accordingly, suppression of
Bcl-2 with siRNA increased the susceptibility of KG1a
and Kasumi-1 cells to DNR-induced apoptosis. These
results provide the first evidence for the ability of curcu-
min to overcome insensitivity to DNR by down-regula-
tion of Bcl-2 in CD34+ AML progenitors. Anti-apoptotic Bcl-2 contributes to the survival and
chemoresistance of quiescent leukemia CD34+ cells [14]. CD34+ AML cells have higher levels of Bcl-2 gene and
protein than CD34- AML cells [6]. DNR-induced apop-
tosis can be blocked by Bcl-2 overexpression in DNR-
sensitive CD34- U937 cells [13]. Conversely, suppression
of Bcl-2 expression with siRNA enhanced DNR-induced
apoptosis in DNR-insensitive CD34+ KG1a and Kasumi-
1 cells. These results suggest that high levels of Bcl-2
expression could contribute to DNR-insensitivity, and
that down-regulation of Bcl-2 by curcumin could be a
molecular mechanism whereby curcumin can overcome
the insensitivity of CD34+ AML cells to DNR. Insensitivity to chemotherapy is a major obstacle to
cancer treatment. CD34+ cell lines display natural resis-
tance to mitoxantrone associated with an absence of
apoptosis [9], giving these immature myeloid leukemia
cells a survival advantage over the more mature leuke-
mia hematopoietic compartment. Curcumin induced
apoptosis in more mature HL-60 AML cells by releasing
cytochrome c and activating caspase-3 [18]. The results
of the present study demonstrated that curcumin
induced apoptosis in both DNR-sensitive U937 cells and
DNR-insensitive KG1a and Kasumi-1 cells via the We further demonstrated that primary CD34+ AML
cells also underwent proliferation inhibition and apopto-
sis with curcumin exposure. This effect was replicated
in 9 individual patient samples representative of differ-
ent French-American-British (FAB) classifications. Furthermore, curcumin also suppressed Bcl-2 expression
and synergistically enhanced DNR cytotoxicity in pri-
mary CD34+ AML cells. These primary cells with differ-
ent FAB classifications represented a broad cross-section
of common AML types, suggesting that down-regulation Rao et al. Conclusion 1. Geller RB, Zahurak M, Hurwitz CA, Burke PJ, Karp JE, Piantadosi S, Civin CI:
Prognostic importance of immunophenotyping in adults with acute
myelocytic leukaemia: the significance of the stem-cell glycoprotein
CD34 (My10). Br J Haematol 1990, 76:340-347. 1. Geller RB, Zahurak M, Hurwitz CA, Burke PJ, Karp JE, Piantadosi S, Civin CI:
Prognostic importance of immunophenotyping in adults with acute
myelocytic leukaemia: the significance of the stem-cell glycoprotein
CD34 (My10). Br J Haematol 1990, 76:340-347. In summary, this study demonstrated a potential new
mechanism whereby curcumin could overcome DNR
insensitivity by down-regulating Bcl-2 in both CD34+
AML cell lines and in primary CD34+ AML cells. Cur-
cumin, either alone or in combination with DNR, could
thus be a potential anti-leukemic agent for the treat-
ment of DNR-insensitive CD34+ AML cells. y
2. Myint H, Lucie NP: The prognostic significance of the CD34 antigen in
acute myeloid leukaemia. Leuk Lymphoma 1992, 7:425-429. y
y
3. Repp R, Schaekel U, Helm G, Thiede C, Soucek S, Pascheberg U, Wandt H,
Aulitzky W, Bodenstein H, Sonnen R, Link H, Ehninger G, Gramatzki M, AML-
SHG Study Group: Immunophenotyping is an independent factor for risk
stratification in AML. Cytometry B Clin Cytom 2003, 53:11-19. y
y
y
4. Tallman MS, Gilliland DG, Rowe JM: Drug therapy for acute myeloid
leukemia. Blood 2005, 106:1154-1163. Discussion
CD3
i i Journal of Translational Medicine 2011, 9:71
http://www.translational-medicine.com/content/9/1/71 Page 14 of 15 Dongfeng Road East, Guangzhou 510060, P.R. China. 5School of Life
Sciences, Sun Yat-Sen University, No. 135 Xingang Xi Road, Guangzhou
510275, P.R. China. of Bcl-2 and induction of apoptosis by curcumin could
be a common death mechanism in CD34+ AML cells. of Bcl-2 and induction of apoptosis by curcumin could
be a common death mechanism in CD34+ AML cells. Several phase I and phase II clinical trials have indi-
cated the potential therapeutic efficacy and lack of toxic
side effects associated with curcumin [28,29]. However,
its poor bioavailability has limited its use for the treat-
ment of cancers outside the gastrointestinal tract [45]. Modern techniques such as the use of synthetic analogs,
derivatives, different formulations and heat-solubilized
curcumin have been explored with the aim of improving
its bioavailability [46-48]; e.g., the water solubility of
curcumin could be increased 12-fold by heating, without
destroying its biological activity [47,48]. Several phase I and phase II clinical trials have indi-
cated the potential therapeutic efficacy and lack of toxic
side effects associated with curcumin [28,29]. However,
its poor bioavailability has limited its use for the treat-
ment of cancers outside the gastrointestinal tract [45]. Modern techniques such as the use of synthetic analogs,
derivatives, different formulations and heat-solubilized
curcumin have been explored with the aim of improving
its bioavailability [46-48]; e.g., the water solubility of
curcumin could be increased 12-fold by heating, without
destroying its biological activity [47,48]. Abbreviations
MAPK
i 11. Suarez L, Vidriales MB, Moreno MJ, Lopez A, Garcia-Larana J, Perez-Lopez C,
Tormo M, Lavilla E, Lopez-Berges MC, de Santiago M, San Miguel JF,
Orfao A, PETHEMA Cooperative Group: Differences in anti-apoptotic and
multidrug resistance phenotypes in elderly and young acute myeloid
leukemia patients are related to the maturation of blast cells. Haematologica 2005, 90:54-59. 11. Suarez L, Vidriales MB, Moreno MJ, Lopez A, Garcia-Larana J, Perez-Lopez C,
Tormo M, Lavilla E, Lopez-Berges MC, de Santiago M, San Miguel JF,
Orfao A, PETHEMA Cooperative Group: Differences in anti-apoptotic and
multidrug resistance phenotypes in elderly and young acute myeloid
leukemia patients are related to the maturation of blast cells. Haematologica 2005, 90:54-59. MAPK: mitogen-activated protein kinase; NF-κB: nuclear factor kappa B;
mTOR: mammalian target of rapamycin; PI3K: phosphoinositide 3-kinase; Bcl-
2: B cell lymphoma 2; IAP: inhibitor of apoptosis protein. Acknowledgements We thank Fucheng Zhang and Huiqiong Lu (Central Lab, Third Affiliated
Hospital, Sun Yat-sen University) for their technical supports. We thank Min
Yan, Jie Xu, Yan Zhao and other members of Liu Lab for technical assistance. This work was supported by a grant from National Nature Science Fund for
Distinguished Young Scholars (30888003 to Q.L.) and National Nature
Science Foundation of China (30873084 to Q.L. and 81000217 to Zi-Jie
Long). 12. Chang H, Salma F, Yi QL, Patterson B, Brien B, Minden MD: Prognostic
relevance of immunophenotyping in 379 patients with acute myeloid
leukemia. Leuk Res 2004, 28:43-48. 13. Kim YH, Park JW, Lee JY, Surh YJ, Kwon TK: Bcl-2 overexpression prevents
daunorubicin-induced apoptosis through inhibition of XIAP and Akt
degradation. Bioche Pharmacol 2003, 66:1779-1786. 14. Konopleva M, Zhao S, Hu W, Jiang S, Snell V, Weidner D, Jackson CE,
Zhang X, Champlin R, Estey E, Reed JC, Andreeff M: The anti-apoptotic
genes Bcl-xl and Bcl-2 are over-expressed and contribute to
chemoresistance of non-proliferating leukaemic CD34+ cells. Brit J
Haematol 2002, 18:521-534. Additional material van-Stijn A, van-der-Pol MA, Kok A, Bontje PM, Roemen GM, Beelen RH,
Ossenkoppele GJ, Schuurhuis GJ: Differences between the CD34+ and
CD34- blast compartments in apoptosis resistance in acute myeloid
leukemia. Haematologica 2003, 88:497-508. 10. van-Stijn A, van-der-Pol MA, Kok A, Bontje PM, Roemen GM, Beelen RH,
Ossenkoppele GJ, Schuurhuis GJ: Differences between the CD34+ and
CD34- blast compartments in apoptosis resistance in acute myeloid
leukemia. Haematologica 2003, 88:497-508. Authors’ contributions JR carried out protein studies, apoptosis analysis, statistical analysis, and
drafted the manuscript. FZ and ZL performed the protein studies, apoptosis
analysis. SH, XW and WZ participated in the statistical analysis. QL and RH
conceived of the study, and participated in its design and coordination. DX
contributed effort in designing, performing, and analyzing the results and
conclusion. All authors read and approved the final manuscript. Additional material 5. Estey E, Dohner H: Acute myeloid leukaemia. Lancet 2006, 368:1894-1907. 6. Shman TV, Fedasenka UU, Savitski VP, Aleinikova OV: CD34+ leukemic
subpopulation predominantly displays lower spontaneous apoptosis and
has higher expression levels of Bcl-2 and MDR1 genes than CD34- cells
in childhood AML. Ann Hematol 2008, 87:353-360. Additional file 1: Figure S1 Curcumin inhibited clonogenic growth. (A) The colonies (containing ≥50) were counted after 14 days by light
microscopy (magnification ×40). (B) Results show numbers of colonies in
the curcumin-treated group expressed as a percentage of number of
colonies in the DMSO-treated group. The graph displays the means ± SD
of three independent experiments. 7. Baer MR, Stewart CC, Dodge RK, Leget G, Sule N, Mrozek K, Schiffer CA,
Powell BL, Kolitz JE, Moore JO, Stone RM, Davey FR, Carroll AJ, Larson RA,
Bloomfield CD: High frequency of immunophenotype changes in acute
myeloid leukemia at relapse: implications for residual disease detection
(Cancer and Leukemia Group B Study 8361). Blood 2001, 97:3574-3580. 7. Baer MR, Stewart CC, Dodge RK, Leget G, Sule N, Mrozek K, Schiffer CA,
Powell BL, Kolitz JE, Moore JO, Stone RM, Davey FR, Carroll AJ, Larson RA,
Bloomfield CD: High frequency of immunophenotype changes in acute
myeloid leukemia at relapse: implications for residual disease detection
(Cancer and Leukemia Group B Study 8361). Blood 2001, 97:3574-3580. Additional file 2: Figure S2 Morphological changes in nuclei in
curcumin-treated cells. (A) KG1a, Kasumi-1 and U937 cells were
incubated with the indicated concentrations of 0, 40, 60, and 80 μM
curcumin for 24 h. Cells were stained with Hoechst 33342 and then
examined under a light microscope. 8. Bailly JD, Muller C, Jaffrezou JP, Demur C, Gassar G, Bordier C, Laurent G:
Lack of correlation between expression and function of P-glycoprotein
in acute myeloid leukemia cell lines. Leukemia 1995, 9:799-807. 9. Bailly JD, Skladanowski A, Bettaieb A, Mansat V, Larsen AK, Laurent G:
Natural resistance of acute myeloid leukemia cell lines to mitoxantrone
is associated with lack of apoptosis. Leukemia 1997, 11:1523-1532. Additional file 3: Table S1 Q value. Q values are shown. Q = 0.85-1.15
indicates simple addition; Q > 1.15 indicates synergism. Additional file 3: Table S1 Q value. Q values are shown. Q = 0.85-1.15
indicates simple addition; Q > 1.15 indicates synergism. 10. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 1 November 2010 Accepted: 19 May 2011
Published: 19 May 2011 Received: 1 November 2010 Accepted: 19 May 2011
Published: 19 May 2011 Author details
1 1Department of Hematology, Third Affiliated Hospital, Sun Yat-sen University,
600 Tianhe Road, Guangzhou 510630, P.R. China. 2Sun Yat-sen Institute of
Hematology, 600 Tianhe Road, Guangzhou 510630, P.R. China. 3Department
of Hematology, First Affiliated Hospital, Sun Yat-sen University, 58
Zhongshan II Road, Guangzhou 510080, P.R. China. 4State Key Laboratory of
Oncology in South China, Cancer Center, Sun Yat-sen University, 651 15. Anand P, Sundaram C, Jhurani S, Kunnumakkara AB, Aggarwal BB:
Curcumin and cancer: an “old-age” disease with an “age-old” solution. Cancer Lett 2008, 267:133-164. Page 15 of 15 Page 15 of 15 Rao et al. Journal of Translational Medicine 2011, 9:71
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Cite this article as: Rao et al.: Curcumin reduces expression of Bcl-2,
leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid
leukemia cell lines and primary sorted CD34+ acute myeloid leukemia
cells. Journal of Translational Medicine 2011 9:71. 29. Dhillon N, Aggarwal BB, Newman RA, Wolff RA, Kunnumakkara AB,
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1 Asou H, Tashiro S, Hamamoto K, Otsuji A, Kita K, Kamada N: Establishment
of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21
chromosome translocation. Blood 1991, 77:2031-2036. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit doi:10.1186/1479-5876-9-71
Cite this article as: Rao et al.: Curcumin reduces expression of Bcl-2,
leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid
leukemia cell lines and primary sorted CD34+ acute myeloid leukemia
cells. Journal of Translational Medicine 2011 9:71. Submit your next manuscript to BioMed Central
and take full advantage of: 32. Notarbartolo M, Poma P, Perri D, Dusonchet L, Cervello M, D’Alessandro N:
Antitumor effects of curcumin, alone or in combination with cisplatin or
doxorubicin, on human hepatic cancer cells. Analysis of their possible
relationship to changes in NF-kB activation levels and in IAP gene
expression. Cancer Lett 2005, 16:53-65. • Convenient online submission 33. Anderson EM, Miller P, Ilsley D, Marshall W, Khvorova A, Stein CA,
Benimetskaya L: Gene profiling study of G3139- and Bcl-2-targeting
siRNAs identifies a unique G3139 molecular signature. Cancer Gene Ther
2006, 13:406-414. 34. Chou TC: Theoretical basis, experimental design, and computerized
simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006, 58:621-681.
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EURASIAN JOURNAL OF LAW, FINANCE AND
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KALIT SO’ZLAR
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haqida soʻz yuritar ekanmiz, bu soʻzning ISSN 2181-2853 Page 123 EURASIAN JOURNAL OF LAW, FINANCE AND
APPLIED SCIENCES Korrupsiya davlatni
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kelajakka ishonch tuygʻularining soʻnishiga
olib keladi. O'z mansab yoki xizmat
mavqeini suiisteʼmol qilish, undan shaxsiy
yoki oʻzgalar manfaatlarini koʻzlab moddiy
yoxud nomoddiy naf olish maqsadida
qonunga xilof ravishda foydalanish jinoiy
javobgarlikka
sabab
boʻladi. Anglaganingizdek, gap korrupsiya haqida
ketmoqda. Bugun jamiyatimizda ushbu
illatga qarshi jiddiy kurashilyapti, uning
oqibatlari
ommaviy
axborot
vositalari
orqali, turli uchrashuvlar, davra suhbatlari,
tadbirlarda
aholiga
tushuntirilishiga
qaramay, afsuski, korrupsiyaning keskin
kamayishiga
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Adubação nitrogenada em milho pelo monitoramento do nível de nitrogênio na planta por meio do clorofilômetro
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Revista Brasileira de Ciência do Solo
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Termos de indexação: Zea mays, leitura SPAD, rendimento de grãos, teor relativo
de clorofila, teor de N. (1) Extraído da Tese de Doutorado, apresentada pelo primeiro autor à Universidade Federal do Rio Grande do Sul – UFRGS. Parci-
almente financiado pela Empresa Pioneer Sementes LTDA. Recebido para publicação em janeiro de 2002 e aprovado em novem-
bro de 2002. (2) Engenheiro-Agrônomo, Syngenta Seeds Ltda. Universidade Federal do Rio Grande do Sul – UFRGS. Departamento de Plantas de
Lavoura. Av. Bento Gonçalves 7712, Caixa Postal 776. CEP 91540-000 Porto Alegre (RS). E-mail: gilberargenta@aol.com;
gilber.argenta@syngenta.com Professor da Faculdade de Agronomia, UFRGS. Bolsista do CNPq. E mail: paulo.silva@vortex.ufrgs.br
(4) Estudante da Faculdade de Agronomia, UFRGS. Bolsista de Iniciação Científica do CNPq. E-mail:planta (3) Professor da Faculdade de Agronomia, UFRGS. Bolsista do CNPq. E-mail: paulo.silva@vortex.ufrgs.br
(4) Estudante da Faculdade de Agronomia UFRGS Bolsista de Iniciação Científica do CNPq E mail:planta Faculdade de Agronomia, UFRGS. Bolsista de Iniciação Científica do CNPq. E-mail:plantas@ufrgs.br da Faculdade de Agronomia, UFRGS. Bolsista do CNPq. E-mail: paulo.silva@vortex.ufrgs.br
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l i
d
i i
Ci
ífi
d
C
il
l (4) Estudante da Faculdade de Agronomia, UFRGS. Bolsista de Iniciação Científica do CNPq. E-mai INTRODUÇÃO filosofia de manejo dos cultivos que poderá
contribuir para evitar a sub ou aplicação excessiva
de fertilizantes nitrogenados. Trata-se de uma
tecnologia de informações que possibilita o
gerenciamento da atividade agrícola, levando-se em
consideração as variabilidades, espacial e temporal,
do solo e da cultura (Fraisse, 1998). Uma abordagem
similar, mas que não utiliza equipamentos
sofisticados, pode ser feita por amostragens pontuais
de características de solo e da planta. É neste
contexto que o monitoramento do nível de N na
planta de milho, pela utilização de curvas de nível
adequado durante alguns estádios específicos do
desenvolvimento vegetativo da cultura, pode
contribuir para a adoção da filosofia de manejo da
agricultura de precisão. Alguns modelos matemáticos foram desenvolvidos
com o objetivo de monitorar o crescimento e o
desenvolvimento da planta de milho (Sinclair &
Muchow, 1995). No entanto, a aplicação destes
modelos em lavoura apresenta limitações, pelo fato
de serem imprevisíveis as condições climáticas que
determinam o crescimento da planta e a disponibili-
dade de N no solo, e, de acordo com Lemaire & Gastal
(1997), pelo pouco conhecimento disponível sobre os
mecanismos básicos que governam o ciclo de N. Considerando a variabilidade do clima e a
necessidade de recomendar a adubação nitrogenada,
esta tem sido, em muitos casos, sub ou superestimada. Assim, quando ela é subestimada, ocorre redução no
rendimento de grãos; quando é superestimada,
diminuem os lucros do agricultor pelo gasto
desnecessário com compra de adubo nitrogenado e
há prejuízos ao meio ambiente, decorrente da
lixiviação de nitrato em condições com excesso de N
disponível (Waskom et al., 1996; Schröder et al., 1998). O monitoramento do nível adequado de N na
planta tem como objetivo diagnosticar a necessidade
ou não da sua aplicação, visto que o uso de altas
doses deste nutriente pode contaminar as águas
superficiais e subterrâneas com nitrato (Waskom et
al., 1996; Varvel et al., 1997; Schröder et al., 2000). Além disso, o uso desta técnica objetiva aumentar a
eficiência do uso de N, visto que a lixiviação deste
nutriente sob forma de nitrato é considerada um
dos principais fatores responsáveis pela sua baixa
eficiência de uso (Raun & Johnson, 1999). Neste
sentido, o monitoramento pode propiciar melhor
sincronismo entre as necessidades deste nutriente
pela cultura e a sua disponibilidade no solo. RESUMO O monitoramento do nível adequado de nitrogênio (N) na planta de milho
tem como objetivo diagnosticar a necessidade ou não de sua aplicação, visto que
o emprego de altas doses deste nutriente pode contaminar as águas superficiais
e subterrâneas com nitrato. O objetivo deste trabalho foi avaliar o teor de
clorofila na folha, medido por meio do clorofilômetro como indicador do nível
de N na planta de milho, em quatro estádios de desenvolvimento. Um experimento
foi realizado no município de Eldorado do Sul, na região fisiográfica da Depressão
Central do estado do Rio Grande do Sul, no ano agrícola de 1999/2000. Os
tratamentos constaram de dois híbridos de milho (Pioneer 32R21 e Premium) e
de oito sistemas de manejo de N em cobertura. As variáveis avaliadas, rendimento
de grãos, teor e acúmulo de N na folha e na planta, nos sistemas monitorados
com o clorofilômetro, não diferiram em relação aos sistemas padrões em que o N
foi aplicado, independentemente das leituras efetuadas. Com o monitoramento
do nível de N na planta do híbrido Pioneer 32R21, houve redução de aplicação
de 50, 100 e 150 kg ha-1 de N, respectivamente, nos sistemas S3, S4 e S5 e, no
híbrido Premium, de 150 kg ha-1 de N, no sistema S5, sem influir no rendimento
de grãos de milho. Portanto, o monitoramento do nível de N na planta de milho
por meio do valor correspondente do teor de clorofila na folha, obtido pelo
clorofilômetro, evidenciou ser eficiente método para separar plantas com
deficiência e com nível adequado deste nutriente. Termos de indexação: Zea mays, leitura SPAD, rendimento de grãos, teor relativo
de clorofila, teor de N. (1) Extraído da Tese de Doutorado, apresentada pelo primeiro autor à Universidade Federal do Rio Grande do Sul – UFRGS. Parci-
almente financiado pela Empresa Pioneer Sementes LTDA. Recebido para publicação em janeiro de 2002 e aprovado em novem-
bro de 2002 (2) Engenheiro-Agrônomo, Syngenta Seeds Ltda. Universidade Federal do Rio Grande do Sul – UFRGS. Departamento de Plantas de
Lavoura. Av. Bento Gonçalves 7712, Caixa Postal 776. CEP 91540-000 Porto Alegre (RS). E-mail: gilberargenta@aol.com;
gilber.argenta@syngenta.com R. Bras. Ci. Solo, 27:109-119, 2003 110 G. ARGENTA et al. Index terms: Zea mays, SPAD reading, grain yield, relative chlorophyll content, nitrogen
concentration. SUMMARY: NITROGEN FERTILIZATION IN MAIZE BY MONITORING THE
PLANT N LEVEL BY A CHLOROPHYLL METER The objective of monitoring the adequate nitrogen (N) content in maize is to determine
when a nutrient application becomes necessary, since the use of high doses of this nutrient
may contaminate superficial and underground waters with nitrate. This experiment was
conducted to evaluate the chlorophyll content in leaves, measured by a chlorophyll meter, as
an indicator of the N level in maize, at four growth stages, in the state of Rio Grande do Sul,
Brazil, during the growing season 1999/2000. Treatments consisted of two maize hybrids
(Pioneer 32R21 and Premium) and eight nitrogen management systems. The tested variables
(grain yield, N level and accumulation in leaf and plant) in the systems monitored by the
chlorophyll meter did not differ in relation to the standard systems, where N was applied
regardless of the readings. Monitoring of the N content in the hybrid Pioneer 32R21, reduced
N applications by 50, 100 and 150 kg ha-1, respectively, in the S3, S4, and S5 systems, and
in the hybrid Premium by 150 kg ha-1 in the system S5, without affecting the maize grain
yield. Therefore, monitoring the N level in maize by the corresponding chlorophyll leaf content
with a portable chlorophyll meter proved to be an efficient method to separate plants with N
deficiency from those with an adequate level of this nutrient. Index terms: Zea mays, SPAD reading, grain yield, relative chlorophyll content, nitrogen
concentration. R. Bras. Ci. Solo, 27:109-119, 2003 ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... 111 Os tratamentos constaram de dois genótipos de
milho (Pioneer 32R21, híbrido simples e de ciclo
superprecoce, e Premium, híbrido simples e de ciclo
precoce) e de oito sistemas (S) de manejo de N em
cobertura. Os sistemas de manejo S1 a S5 represen-
tam doses de N em cobertura, respectivamente, 0 ,
70, 175, 350 e 525 kg ha-1. As doses de N foram
aplicadas em quatro épocas de desenvolvimento das
plantas. No estádio de três a quatro folhas, foram
aplicadas as doses de N de 10, 25, 50 e 75 kg ha-1,
correspondendo, respectivamente, aos sistemas S2,
S3, S4 e S5, considerados como sistemas padrões. O
restante do N foi aplicado em três doses iguais, nos
estádios de seis a sete folhas, 10 a 11 folhas
expandidas e de espigamento. Outros três sistemas
equivalentes, respectivamente aos sistemas S3, S4
e S5, foram os tratamentos monitorados, em que as
doses de N somente foram aplicadas quando as
leituras obtidas no clorofilômetro eram inferiores aos
valores estabelecidos como adequados nos respectivos
estádios de desenvolvimen-to da planta. As leituras
no clorofilômetro correspondentes ao nível adequado
de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0,
respectivamente, para os estádios de três a quatro
folhas, seis a sete folhas, 10 a 11 folhas expandidas
e de espigamento. Maiores detalhes de como foram
determinados estes valores estão descritos em
Argenta (2001). correlação com rendimento de grãos e aceitável nível
de exatidão. No entanto, por serem laboratoriais,
revelam desvantagens de despender tempo e trabalho,
envolver despesas com coleta, processamento e
análise de amostras e, principalmente, não
possibilitar a correção da deficiência de N na planta
no mesmo ano agrícola, servindo apenas como
critério indicativo para os próximos anos (Argenta,
2001). O desenvolvimento do medidor portátil de
clorofila, equipamento que permite medições
instantâneas do valor correspondente ao seu teor
na folha, constitui alternativa promissora para
avaliação do nível de N nas plantas (Argenta et al.,
2001a). Alguns pesquisadores evidenciaram relação
entre leitura do clorofilômetro e teor de clorofila na
folha (Yadava, 1986; Marquard & Tipton, 1987;
Dwyer et al., 1995; Argenta et al., 2001b) e entre
teor de clorofila na folha e teor de N na planta (Smeal
& Zhang, 1994; Argenta et al., 2001b). ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... Em outro
trabalho, em que se testaram características da
planta (teor e acúmulo de N, leitura correspondente
ao teor de clorofila na folha, avaliada com
clorofilômetro, produção de matéria seca e área
foliar) como indicadores do nível de N na planta de
milho, foi constatado que a leitura no clorofilômetro
foi o melhor indicador do nível de N na planta dentre
as características avaliadas (Argenta et al., 2001c). Argenta (2001) determinou que, para diagnóstico do
nível de N na planta de milho, as leituras no
clorofilômetro acima de 45,4, 52,1, 55,3 e 58,0,
respectivamente, para os estádios de três a quatro
folhas, seis a sete folhas, 10 a 11 folhas e de
espigamento, representam nível adequado de N. Os híbridos de milho foram semeados no dia 18
de outubro de 1999 em semeadura direta, em
sucessão ao consórcio de aveia preta e ervilhaca
comum, com espaçamento entre linhas de 0,7 m e
densidade de 75.000 plantas ha-1. O delineamento
experimental utilizado foi de blocos casualizados com
parcelas subdivididas, com quatro repetições. Os
híbridos foram locados nas parcelas principais,
enquanto os sistemas de manejo de N, nas
subparcelas. A análise do solo, realizada antes da
instalação do experimento, indicou os seguintes
valores: teor de argila de 28 dag kg-1; pH 5,2; teores
de fósforo, potássio e matéria orgânica de 16,
134 mg dm-3 e 2,6 dag kg-1, respectivamente. Por
ocasião da semeadura, foi efetuada adubação em
linha com 30 kg ha-1 de N, 120 kg ha-1 de P2O5 e
120 kg ha-1 de K2O. O objetivo deste experimento foi avaliar o teor
de clorofila na folha, medido por meio do
clorofilômetro, como método indicador do nível de N
na planta de milho, em quatro estádios de
desenvolvimento. INTRODUÇÃO A sub, ou superestimação, da dose de N a ser
utilizada ocorre rotineiramente no sistema
tradicional de recomendação de adubação (aplicação
de 1/3 na semeadura e o restante em cobertura), pelo
fato de serem adotados conjuntos de práticas
culturais em lavouras sem considerar suas
particularidades de desuniformidade. Por esse
motivo, haverá áreas (manchas) em que a adubação
aplicada estará abaixo da necessidade das plantas
(subdose) e outras em que ela estará acima da
necessidade. Alguns métodos de previsão da necessidade de
N durante o desenvolvimento vegetativo da planta
de milho têm sido propostos (Binford et al., 1992;
Sims et al., 1995). Baseados em testes de solo e em
análises laboratoriais de amostras de tecido, tais
métodos têm a vantagem de apresentar boa A agricultura de precisão, dentre outras
finalidades, está sendo proposta como uma nova R. Bras. Ci. Solo, 27:109-119, 2003 RESULTADOS E DISCUSSÃO Nos estádios de três a quatro folhas e de seis a
sete folhas de milho completamente desenvolvidas,
os valores de leitura obtidos no clorofilômetro
estiveram abaixo dos considerados adequados,
respectivamente, 45,4 e 52,1, em todos os sistemas
de manejo de N e nos dois híbridos (Quadro 1). Este
resultado indica que as doses de N aplicadas na
semeadura (30 kg ha-1) e da primeira dose de
cobertura (10 a 75 kg ha-1) não foram suficientes
para suprir as necessidades das plantas até esses
estádios. Possivelmente, o alto rendimento de
matéria seca da parte aérea das coberturas de solo
no inverno em consórcio (4,5 t ha-1) fez com que os
microrganismos quimiorganotrópicos que atuam na
decomposição do material orgânico se multiplicassem
rapidamente, assimilando carbono e produzindo CO2 As plantas e as folhas individuais utilizadas nas
leituras foram secas em estufa a ± 60 ºC até atingirem
peso constante. A quantidade de matéria seca obtida
por amostra foi dividida por cinco, para determinar
a produção de matéria seca por planta e por folha
avaliada. O teor de N foi determinado de acordo
com método descrito em Tedesco et al. (1995). As
quantidades de N acumuladas por planta e por folha
foram calculadas, multiplicando-se, respectivamente,
a produção de matéria seca da parte aérea por planta
e por folha avaliada pelo teor de N. O rendimento de grãos de milho foi estimado,
extrapolando-se a produção colhida na área útil das
subparcelas (7 m2) para um hectare, corrigindo-se a
umidade para 130 dag kg-1. Quadro 1. Leitura do clorofilômetro (leitura SPAD) na folha de dois híbridos de milho, considerando oito
sistemas de manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado
do Sul (RS), 1999/2000 Quadro 1. Leitura do clorofilômetro (leitura SPAD) na folha de dois híbridos de milho, considerando oito
sistemas de manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado
do Sul (RS), 1999/2000 Quadro 1. Leitura do clorofilômetro (leitura SPAD) na folha de dois híbridos de milho, considerando oito
sistemas de manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado
do Sul (RS), 1999/2000
(1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. G. ARGENTA et al. G. ARGENTA et al. estádio de espigamento, as leituras foram feitas na
folha-índice (primeira abaixo da espiga). Os dados obtidos foram submetidos à análise de
variância pelo teste F. Quando significativos,
compararam-se as médias pelo teste de Duncan
(P ≤ 0,05). As leituras no medidor de clorofila (duas por
folha) foram feitas em pontos situados na metade a
dois terços do comprimento da folha, a partir da base,
e a 2 cm de uma das margens da folha. Após a
leitura, as folhas amostradas foram coletadas em
separado do resto da planta para determinação do
teor e do acúmulo de N. MATERIAL E MÉTODOS O experimento foi realizado em campo, na
Estação Experimental Agronômica da Universidade
Federal do Rio Grande do Sul, localizada no
município de Eldorado do Sul, região fisiográfica da
Depressão Central, do estado do Rio Grande do Sul,
na estação de crescimento 1999/00. O clima da região
é classificado, segundo Köppen, como subtropical
úmido, situado na transição entre os tipos
fundamentais cfa1 (isoterma anual inferior a 18 ºC)
e cfa2 (isoterma anual superior a 18 ºC) (Moreno,
1961). As temperaturas médias (anual, máxima e
mínima) são de 19,6, 24,3 e 14,8 ºC, respectivamente
(IPAGRO, 1989). O solo da área experimental é
classificado como Argissolo Vermelho distrófico típico
(EMBRAPA, 1999). As determinações feitas foram: leitura
correspondente ao teor de clorofila na folha, avaliada
com o clorofilômetro modelo Minolta SPAD-502,
produção de matéria seca da parte aérea por planta
e da folha avaliada, teor de N total no tecido e
quantidade de N acumulada por planta e por folha
avaliada. As determinações foram realizadas nos
estádios de três a quatro folhas, seis a sete folhas e
de 10 a 11 folhas completamente desenvolvidas e no
espigamento, utilizando cinco plantas e cinco folhas
por subparcela. Nos estádios vegetativos, as leituras
com medidor de clorofila foram feitas na 3ª, 6ª e
9ª folhas totalmente expandidas, correspondendo,
respectivamente, aos estádios de três a quatro folhas,
seis a sete folhas e 10 a 11 folhas expandidas. No R. Bras. Ci. Solo, 27:109-119, 2003 112 RESULTADOS E DISCUSSÃO As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Valor de leitura do clorofilômetro superior ao estabelecido como equivalente ao nível adequado de N na planta. Sistema de manejo de N(1)
S1
S2
S3
S4
S5
Híbrido
Padrão
Padrão
Padrão
Monitorado
Padrão
Monitorado
Padrão
Monitorado
Leitura SPAD
Estádio de 3-4 folhas
P 32R21
38,4
--(2)
--
--
--
--
--
--
Premium
35,7
--
--
--
--
--
--
--
Estádio de 6-7 folhas
P 32R21
45,4
44,9
--(2)
46,2
--
47,6
--
47,5
Premium
44,4
45,4
--
47,2
--
47,5
--
48,5
Estádio de 10-11 folhas
P 32R21
48,2
53,1
--(2)
55,8(3)
--
57,4(3)
--
56,5(3)
Premium
49,6
50,7
--
52,9
--
54,4
--
55,3(3)
Estádio de espigamento
P 32R21
41,1
45,5
50,8
47,7
51,8
49,6
51,4
51,3
Premium
40,8
44,7
46,8
47,6
46,7
46,2
50,0
49,2 ( ) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. RESULTADOS E DISCUSSÃO O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Valor de leitura do clorofilômetro superior ao estabelecido como equivalente ao nível adequado de N na planta. Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... Houve efeito significativo de híbridos somente para
teor de N na planta, sendo 30 % maior no híbrido
Premium do que no Pioneer 32R21 (Quadro 4). Avaliando o efeito de sistemas, constatou-se
resposta similar à obtida para teor de N na 9ª folha
para estas três variáveis. O acúmulo de N na 9ª folha
e o teor e o acúmulo de N na planta (estádio10-
11 folhas) foram superiores nos sistemas
monitorados em relação aos sistemas S1 e S2
padrões, com exceção do teor de N na planta para o
qual não houve diferença entre o sistema S3
monitorado e S2 padrão. Os valores no sistema
padrão S2 somente foram similares aos do sistema
S1 para teor de N na planta; para as variáveis
acúmulo de N na 9ª folha e na planta, os valores
foram superiores. No estádio de espigamento, os valores de leitura
do clorofilômetro foram inferiores aos considerados
adequados (leitura SPAD 58,0), em todos os sistemas
de manejo de N e nos dois híbridos. Tais resultados
refletem o nível insuficiente de N nas plantas, ou
seja, todos os tratamentos testados requeriam a
suplementação de adubação nitrogenada. Para
alguns autores, a aplicação de N neste estádio é
pouco eficiente, pois a maior demanda por este
nutriente ocorre cerca de duas a três semanas antes
do florescimento, ou seja, época em que cerca de 95 %
do N total da planta já havia sido absorvido (Muzilli
& Oliveira, 1992; Plénet & Cruz, 1997). No estádio de espigamento, para teor e acúmulo
de N na folha-índice, não houve diferença
significativa entre híbridos (Quadros 2 e 3). Com
relação aos sistemas de manejo de N, constatou-se
que os maiores teores de N na folha foram obtidos
nos sistemas S5, padrão e monitorado, e S4 padrão
não diferindo do sistema S4 monitorado. Os menores
teores foram verificados no sistema S1 padrão. Nos
demais sistemas, os valores foram intermediários. Na média de sistemas, os teores de N na folha-índice
variaram de 1,40 a 2,02 dag kg-1. Segundo alguns
pesquisadores, a concentração crítica de N na folha-
índice de milho varia de 2,1 a 3,6 dag kg-1 (Roberts
& Rhee, 1993; Soltanpour et al., 1995), dependendo
de variação dentro e entre locais e anos. Portanto, o
teor de N determinado na folha-índice neste trabalho
ficou abaixo do considerado crítico. ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... 113 em grandes quantidades (Argenta & Silva, 1999). Paralelamente à decomposição do material orgânico
(carbono), os microrganismos necessitam assimilar
N. Como conseqüência, o nitrato e o amônio presentes
no solo praticamente desaparecem (Victoria et al.,
1992). quantidades de N acumuladas na 6ª folha e na planta
foram 15 e 7 % superiores no híbrido Pioneer 32R21
em relação ao Premium (Quadros 3 e 5, respectiva-
mente). Com relação aos efeitos de sistemas de
manejo de N, apenas o teor de N na planta variou,
sendo maior nos sistemas S2 padrão e S3 e S4
monitorados do que nos sistemas S1 padrão e S5
monitorado (Quadro 4). No estádio de 10 a 11 folhas, houve resposta
diferencial entre híbridos para leituras no
clorofilômetro. Nos três sistemas monitorados, os
valores da leitura no clorofilômetro no híbrido
Pioneer 32R21 foram superiores aos considerados
adequados (leitura SPAD de 55,3) (Quadro 1). No
híbrido Premium, apenas no sistema S5 monitorado
os valores da leitura foram superiores aos
considerados adequados. Nos sistemas S3 e S4
monitorados, os valores estiveram abaixo, porém,
muito próximos dos considerados adequados. Nos
sistemas padrões S1 e S2, registraram-se leituras
no clorofilômetro abaixo das consideradas ideais, nos
dois híbridos. Portanto, neste estádio, nos sistemas
monitorados S3, S4 e S5 para o híbrido Pioneer 32R21
não foi aplicado N em cobertura, representando
redução nas doses de N a serem aplicadas,
respectivamente, de 50, 100 e 150 kg ha-1. No híbrido
Premium, apenas no sistema S5 não foi aplicada
adubação nitrogenada em cobertura, eqüivalendo à
redução de 150 kg ha-1 de N. No estádio de 10 a 11 folhas de milho, foi
significativa a interação entre híbridos e sistemas
de manejo de N para teor de N na 9ª folha avaliada
(Quadro 2). Apenas nos sistemas S2 padrão e S4
monitorado não houve diferença entre híbridos. Nos
demais, o teor de N na 9ª folha foi maior no híbrido
Premium do que no Pioneer 32R21. Nos dois
híbridos, o teor de N na 9ª folha foi maior nos
sistemas monitorados em relação aos sistemas S1 e
S2 padrões. Para acúmulo de N na 9ª folha, e teor e
acúmulo de N na planta, no estádio de 10 a 11 folhas,
a interação não foi significativa (Quadros 3, 4 e 5). RESULTADOS E DISCUSSÃO Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Valor de leitura do clorofilômetro superior ao estabelecido como equivalente ao nível adequado de N na planta. R. Bras. Ci. Solo, 27:109-119, 2003 ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... No entanto, Rajcan & Tollenaar (1999) verificaram
que 60 e 40 % do N presente nos grãos de milho,
respectivamente, para um híbrido moderno e um
antigo, foram absorvidos após o espigamento. Segundo estes autores, a continuidade de absorção
de N durante o período de enchimento de grãos
implica menor remobilização deste nutriente de
órgãos vegetativos, resultando em aumento da
duração da área foliar e prolongamento do período
de acúmulo de matéria seca. Estas duas caracterís-
ticas são importantes, pois estão associadas a altos
rendimentos de grãos de milho (Moll et al., 1994). Com relação ao teor e acúmulo de N na 3ª folha
(Quadros 2 e 3) e ao teor e acúmulo de N na planta
(Quadros 4 e 5), no estádio de três a quatro folhas,
não houve diferença significativa entre híbridos de
milho. No estádio de seis a sete folhas, os teores de
N na 6ª folha e na parte aérea da planta também
não variaram entre híbridos. No entanto, as Este resultado reforça o obtido de leituras
efetuadas com clorofilômetro na folha-índice em que
foi constatado que as plantas estavam com nível de
N abaixo do considerado adequado (Quadro 1). Para
N acumulado na folha-índice, o sistema que
proporcionou maior acúmulo foi o S5 padrão, não R. Bras. Ci. Solo, 27:109-119, 2003 114 G. ARGENTA et al. Quadro 2. Teor de nitrogênio (N) na folha de dois híbridos de milho, considerando oito sistemas de manejo
de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado do Sul (RS), 1999/2000
Sistema de manejo de N(1)
S1
S2
S3
S4
S5
Híbrido
Padrão
Padrão
Padrão
Monitorado
Padrão
Monitorado
Padrão
Monitorado
Média
Estádio de 3-4 folhas, dag kg-1 de N na 3ª folha
P 32R21
3,86
--(2)
--
--
--
--
--
--
3,86ns
Premium
4,04
--
--
--
--
--
--
--
4,04
Estádio de 6-7 folhas, dag kg-1 de N na 6ª folha
P 32R21
3,07
2,45
--(2)
2,49
--
2,52
--
2,79
2,66ns
Premium
2,16
2,42
--
2,64
--
2,54
--
2,45
2,44
Média
2,62ns
2,44
2,57
2,53
2,62
C.V. (%)
Sistema de manejo de N = 13,5; Híbrido = 8,4
Estádio de 10-11 folhas, dag kg-1 de N na 9ª folha
P 32R21
1,72 bC(3)
2,21 aB
--(2)
2,48 bA
--
2,48 aA
--
2,42 bA
Premium
2,03 aC
2,08 aC
--
2,63 aA
--
2,56 aA
--
2,80 aA
C.V. ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). e S3 monitorado, enquanto o menor foi verificado no
S4 padrão. diferindo dos sistemas S5 monitorado e S4 padrão,
o menor acúmulo ocorreu no sistema S1 e os demais
sistemas tiveram comportamento intermediário. Para acúmulo de N na planta, no estádio de
espigamento, também foi significativa a interação
entre híbridos e sistemas de manejo de N (Quadro 5). O híbrido Premium acumulou maior quantidade de
N nos sistemas S1, S2 e S3 padrões e S3 monitorado
do que o Pioneer 32R21. Nos demais sistemas, não
houve diferença entre híbridos. No híbrido P 32R21,
os sistemas que proporcionaram maior acúmulo de
N na planta foram o S5 padrão e o S5 monitorado,
enquanto o menor acúmulo ocorreu no sistema S1
padrão. No híbrido Premium, os sistemas que
proporcionaram maior acúmulo de N na planta
foram os padrões S4 e S5 e S4 e S5 monitorados,
enquanto os menores valores ocorreram nos sistemas
S3 padrão e S3 monitorado, não diferindo do S1
padrão. Para teor de N na planta, no estádio de
espigamento, foi significativa a interação entre
híbridos e sistemas de manejo de N (Quadro 4). O
teor de N na planta do híbrido Pioneer 32R21 foi
maior do que no Premium somente no sistema S5
monitorado. R. Bras. Ci. Solo, 27:109-119, 2003 ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... (%)
Sistema de manejo de N = 12,5; Híbrido = 3,5
Estádio de espigamento, dag kg-1 de N na folha-índice
P 32R21
1,31
1,59
1,79
1,68
1,92
1,90
2,06
1,93
1,77ns
Premium
1,49
1,78
1,87
1,89
2,13
1,98
1,98
2,02
1,89
Média
1,40 D
1,69 C
1,83 B
1,79 BC
2,03 A
1,94 AB
2,02 A
1,98 A
C.V. (%)
Sistema de manejo de N = 10,4; Híbrido = 4,2
(1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). Quadro 2. Teor de nitrogênio (N) na folha de dois híbridos de milho, considerando oito sistemas de manejo
de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado do Sul (RS), 1999/2000 nitrogênio (N) na folha de dois híbridos de milho, considerando oito sistemas de manejo
o estádios de desenvolvimento da planta. ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... EEA/UFRGS, Eldorado do Sul (RS), 1999/2000 (1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). (1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... Nos sistemas padrões S1 e S2, o teor
de N foi superior no híbrido Premium em relação ao
Pioneer 32R21. Nos demais sistemas, não houve
diferença entre híbridos. No híbrido P 32R21, o
sistema de manejo que proporcionou maior teor de
N na planta foi o S5 padrão, não diferindo do S5
monitorado, enquanto o menor foi obtido no sistema
S1 padrão. No híbrido Premium, o sistema que
proporcionou maior teor de N na planta foi o S1
padrão, não diferindo dos sistemas S2 e S5 padrões ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... 115 Quadro 3. Nitrogênio (N) acumulado na folha de dois híbridos de milho, considerando oito sistemas de
manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado do Sul (RS),
1999/2000
Sistema de manejo de N(1)
S1
S2
S3
S4
S5
Híbrido
Padrão
Padrão
Padrão
Monitorado
Padrão
Monitorado
Padrão
Monitorado
Média
Estádio de 3-4 folhas, mg de N na 3ª folha
P 32R21
10
--(2)
--
--
--
--
--
--
10ns
Premium
8
--
--
--
--
--
--
--
8
Estádio de 6-7 folhas, mg de N na 6ª folha
P 32R21
30
22
--(2)
26
--
28
--
24
26 a(3)
Premium
21
21
--
21
--
22
--
22
22 b
Média
25ns
22
24
25
23
C.V. (%)
Sistema de manejo de N = 22,1; Híbrido = 8,0
Estádio de 10-11 folhas, mg de N na 9ª folha
P 32R21
48
64
--(2)
81
--
74
--
71
68ns
Premium
44
47
--
79
--
67
--
79
68
Média
46 C
56 B
80 A
71 A
75 A
C.V. (%)
Sistema de manejo de N = 19,2; Híbrido = 7,3
Estádio de espigamento, mg de N na folha-índice
P 32R21
50
67
78
67
80
82
101
87
77ns
Premium
63
78
85
89
102
89
92
96
87
Média
57 E
72 D
82 BCD
77 CD
91 AB
86 BC
96 A
92 AB
C.V. (%)
Sistema de manejo de N = 17,2; Híbrido = 6,9
(1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula na coluna, para comparação de híbridos e, seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). Quadro 3. Nitrogênio (N) acumulado na folha de dois híbridos de milho, considerando oito sistemas de
manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado do Sul (RS),
1999/2000
Sistema de manejo de N(1)
S1
S2
S3
S4
S5
Híbrido
Padrão
Padrão
Padrão
Monitorado
Padrão
Monitorado
Padrão
Monitorado
Média
Estádio de 3-4 folhas, mg de N na 3ª folha
P 32R21
10
--(2)
--
--
--
--
--
--
10ns
Premium
8
--
--
--
--
--
--
--
8
Estádio de 6-7 folhas, mg de N na 6ª folha
P 32R21
30
22
--(2)
26
--
28
--
24
26 a(3)
Premium
21
21
--
21
--
22
--
22
22 b
Média
25ns
22
24
25
23
C.V. (%)
Sistema de manejo de N = 22,1; Híbrido = 8,0
Estádio de 10-11 folhas, mg de N na 9ª folha
P 32R21
48
64
--(2)
81
--
74
--
71
68ns
Premium
44
47
--
79
--
67
--
79
68
Média
46 C
56 B
80 A
71 A
75 A
C.V. ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... (%)
Sistema de manejo de N = 19,2; Híbrido = 7,3
Estádio de espigamento, mg de N na folha-índice
P 32R21
50
67
78
67
80
82
101
87
77ns
Premium
63
78
85
89
102
89
92
96
87
Média
57 E
72 D
82 BCD
77 CD
91 AB
86 BC
96 A
92 AB
C.V. (%)
Sistema de manejo de N = 17,2; Híbrido = 6,9
(1) Quadro 3. Nitrogênio (N) acumulado na folha de dois híbridos de milho, considerando oito sistemas de
manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado do Sul (RS),
1999/2000 io (N) acumulado na folha de dois híbridos de milho, considerando oito sistemas de
m quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado do Sul (RS), (1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula na coluna, para comparação de híbridos e, seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). G. ARGENTA et al. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). Quadro 4. Teor de nitrogênio (N) na parte aérea por planta de dois híbridos de milho, considerando oito
sistemas de manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado
do Sul (RS), 1999/2000
Sistema de manejo de N(1)
S1
S2
S3
S4
S5
Híbrido
de milho
Padrão
Padrão
Padrão
Monitorado
Padrão
Monitorado
Padrão
Monitorado
Média
Estádio de 3-4 folhas, dag kg-1
P 32R21
3,50
--(2)
--
--
--
--
--
--
3,50ns
Premium
3,45
--
--
--
--
--
--
--
3,54
Estádio de 6-7 folhas, dag kg-1
P 32R21
2,63
2,66
--(2)
3,15
--
2,47
--
2,77
2,74ns
Premium
2,40
2,57
--
2,65
--
2,86
--
2,21
2,54
Média
2,52 B
2,62 A
2,90 A
2,86 A
2,49 B
C.V. (%)
Sistema de manejo de N = 15,2; Híbrido = 14,5
Estádio de 10-11 folhas, dag kg-1
P 32R21
1,38
1,70
--(2)
2,09
--
2,11
--
2,13
1,79 b(3)
Premium
2,32
2,08
--
2,14
--
2,31
--
2,77
2,32 a
Média
1,85 C
1,89 BC
2,12 AB
2,21 AB
2,45 A
C.V. (%)
Sistema de manejo de N = 20,9; Híbrido =13,1
Estádio de espigamento, dag kg-1
P 32R21
0,72 bE
0,94 bD
1,18 aBC
1,17 aBC
0,97 aCD
1,11 aBC
1,48 aA
1,33 aAB
Premium
1,43 aA
1,34 aAB
1,20 aBC
1,31 aABC
1,03 aD
1,22 aCD
1,30 aABC
1,11 bCD
C.V. (%)
Sistema de manejo de N = 16,7; Híbrido = 6,6 nitrogênio (N) na parte aérea por planta de dois híbridos de milho, considerando oito
anejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado
99/2000 (1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. G. ARGENTA et al. G. ARGENTA et al. sistemas pode diminuir o potencial de contaminação
ambiental pela menor lixiviação de nitrato (Waskom
et al., 1996; Varvel et al., 1997; Schröder et al., 2000)
e aumentar a eficiência de seu uso (Raun & Johnson,
1999). Estes resultados evidenciam que os valores
de leitura no clorofilômetro utilizados para
monitorar o nível de N em milho foram adequados
para separar as plantas com deficiência e com nível
adequado deste nutriente. para recomendação mais exata do manejo de N na
cultura do milho, para maximizar o rendimento de
grãos e diminuir os custos de produção e o impacto
ambiental, dado o excesso de adubação nitrogenada
utilizada. O método proposto para monitorar o nível de N
nas plantas de milho pela leitura do teor relativo de
clorofila demonstra grande aplicabilidade pelo
produtor, mesmo com a limitação de não predizer a
quantidade exata de N necessária para ser aplicada. O método permite fazer um diagnóstico da lavoura
em poucos minutos, fornecendo informações
importantes para a tomada de decisão. O valor da
leitura, quando estiver acima do correspondente ao
nível adequado no estádio avaliado, significa que não
vale a pena aplicar N, pois a planta não está
necessitando deste nutriente naquele momento. Por
outro lado, quando estiver abaixo do considerado Embora seja eficiente para indicar a necessidade
de adubação nitrogenada, a leitura no clorofilômetro
possui a limitação de não quantificar a dose a ser
aplicada. Por outro lado, indicadores de solo que
são melhores para predizer a quantidade de N a ser
aplicada revelam a limitação de não serem precisos
para predizer a necessidade deste nutriente pelas
plantas. Assim, evidencia-se a necessidade de se
integrar o uso de indicadores de solo e de planta Quadro 4. Teor de nitrogênio (N) na parte aérea por planta de dois híbridos de milho, considerando oito
sistemas de manejo de N, em quatro estádios de desenvolvimento da planta. G. ARGENTA et al. EEA/UFRGS, Eldorado
do Sul (RS), 1999/2000
Sistema de manejo de N(1)
S1
S2
S3
S4
S5
Híbrido
de milho
Padrão
Padrão
Padrão
Monitorado
Padrão
Monitorado
Padrão
Monitorado
Média
Estádio de 3-4 folhas, dag kg-1
P 32R21
3,50
--(2)
--
--
--
--
--
--
3,50ns
Premium
3,45
--
--
--
--
--
--
--
3,54
Estádio de 6-7 folhas, dag kg-1
P 32R21
2,63
2,66
--(2)
3,15
--
2,47
--
2,77
2,74ns
Premium
2,40
2,57
--
2,65
--
2,86
--
2,21
2,54
Média
2,52 B
2,62 A
2,90 A
2,86 A
2,49 B
C.V. (%)
Sistema de manejo de N = 15,2; Híbrido = 14,5
Estádio de 10-11 folhas, dag kg-1
P 32R21
1,38
1,70
--(2)
2,09
--
2,11
--
2,13
1,79 b(3)
Premium
2,32
2,08
--
2,14
--
2,31
--
2,77
2,32 a
Média
1,85 C
1,89 BC
2,12 AB
2,21 AB
2,45 A
C.V. (%)
Sistema de manejo de N = 20,9; Híbrido =13,1
Estádio de espigamento, dag kg-1
P 32R21
0,72 bE
0,94 bD
1,18 aBC
1,17 aBC
0,97 aCD
1,11 aBC
1,48 aA
1,33 aAB
Premium
1,43 aA
1,34 aAB
1,20 aBC
1,31 aABC
1,03 aD
1,22 aCD
1,30 aABC
1,11 bCD
C.V. (%)
Sistema de manejo de N = 16,7; Híbrido = 6,6
(1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... ns =
não-significativo (P ≤ 0,05). manejo que proporcionou menor rendimento de
grãos foi o S1. Os maiores rendimentos de grãos
foram obtidos nos sistemas S4 padrão e S5
monitorado, porém sem se diferenciarem do sistema
S4 monitorado. Pode-se verificar que o rendimento
de grãos nos sistemas monitorados não diferiu dos
seus sistemas padrões, com exceção do S5
monitorado, cujo rendimento foi superior ao do S5
padrão. Analisando, conjuntamente, o teor e acúmulo de
N na folha-índice e o teor e acúmulo de N na planta
de milho, no estádio de espigamento, verifica-se que
os valores obtidos foram similares entre os sistemas
de manejo de N padrões e monitorados para as
quatro variáveis (Quadros 2, 3, 4 e 5), indicando que
foi correta a decisão de não se aplicar N nos
tratamentos monitorados, quando os valores da
leitura no clorofilômetro estavam acima do
considerado adequado. Portanto, com o monitoramento do nível de N na
planta do híbrido Pioneer 32R21, houve redução de
aplicação de 50, 100 e 150 kg ha-1 de N,
respectivamente, nos sistemas S3, S4 e S5 e, no
híbrido Premium, de 150 kg ha-1 de N no sistema
S5, sem influir no rendimento de grãos de milho. Além deste fato, a menor aplicação de N nestes O rendimento de grãos de milho variou de acordo
com os híbridos e sistemas de manejo de N, não sendo
constatada interação entre os fatores. O rendimento
de grãos do híbrido Pioneer 32R21 foi 30 % superior
ao obtido pelo híbrido Premium, na média dos
sistemas de manejo de N (Quadro 6). O sistema de R. Bras. Ci. Solo, 27:109-119, 2003 116 G. ARGENTA et al. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete folhas,
10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5 foram os
tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro eram inferiores
ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível adequado de N
utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas, 10 a 11 folhas
e de espigamento.(2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas, por serem as
doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos sistemas
padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). Quadro 6. Rendimento de grãos de dois híbridos de milho, considerando oito sistemas de manejo de N. EEA/UFRGS, Eldorado do Sul (RS), 1999/2000
(1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura, respectivamente de, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5 foram
os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro eram
inferiores ao estabelecido nos respectivos estádios de desenvolvimento. G. ARGENTA et al. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5
foram os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro
eram inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível
adequado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento. (2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas,
por serem as doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos
sistemas padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). R. Bras. Ci. Solo, 27:109-119, 2003 ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... 117 Quadro 5. Nitrogênio (N) acumulado na parte aérea por planta de dois híbridos de milho, considerando
oito sistemas de manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado
do Sul (RS), 1999/2000
Sistema de manejo de N(1)
S1
S2
S3
S4
S5
Híbrido
Padrão
Padrão
Padrão
Monitorado
Padrão
Monitorado
Padrão
Monitorado
Média
Estádio de 3-4 folhas (mg/planta de N)
P 32R21
50
--(2)
--
--
--
--
--
--
50ns
Premium
44
--
--
--
--
--
--
--
44
Estádio de 6-7 folhas (mg/planta de N)
P 32R21
179
223
--(2)
257
--
209
--
212
214 a(3)
Premium
174
220
--
201
--
200
--
188
199 b
Média
177ns
222
229
205
200
C.V. G. ARGENTA et al. Nitrogênio (N) acumulado na parte aérea por planta de dois híbridos de milho, considerando
oito sistemas de manejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado
do Sul (RS), 1999/2000 (N) acumulado na parte aérea por planta de dois híbridos de milho, considerando
anejo de N, em quatro estádios de desenvolvimento da planta. EEA/UFRGS, Eldorado
2000 (1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete folhas,
10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5 foram os
tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro eram inferiores
ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível adequado de N
utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas, 10 a 11 folhas
e de espigamento.(2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas, por serem as
doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos sistemas
padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). (1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. G. ARGENTA et al. (%)
Sistema de manejo de N = 21,7; Híbrido = 19,7
Estádio de 10-11 folhas (mg/planta de N)
P 32R21
490
729
--(2)
648
--
899
--
1012
756ns
Premium
685
674
--
839
--
991
--
912
820
Média
588 C
702 B
744 A
945 A
962 A
C.V. (%)
Sistema de manejo de N = 23,4; Híbrido = 18,5
Estádio de espigamento (mg/planta de N)
P 32R21
814 bC
1196 bB
1462 bB
1314 bB
1290 aB
1363 aB
1825 aA
1834 aA
Premium
1524 aBC
1815 aAB
1479 aC
1383 aC
1856 aA
1852 aA
1946 aA
1928 aA
C.V. (%)
Sistema de manejo de N = 20,5; Híbrido = 10,9
(1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura de, respectivamente, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete folhas,
10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5 foram os
tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro eram inferiores
ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível adequado de N
utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas, 10 a 11 folhas
e de espigamento.(2) No estádio de três a quatro folhas expandidas, apenas as plantas do sistema S1 foram avaliadas, por serem as
doses de N todas iguais até este estádio. Nos estádios de seis a sete folhas e de 10 a 11 folhas expandidas, as plantas dos sistemas
padrões S3, S4 e S5 não foram avaliadas, pois, até estes estádios, estes tratamentos foram similares aos sistemas monitorados. (3) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas pela mesma letra maiúscula, na
linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo teste de Duncan, (P ≤ 0,05). ns =
não-significativo (P ≤ 0,05). Quadro 5. G. ARGENTA et al. As leituras no clorofilômetro correspondentes ao nível ade-
quado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento.(2) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas
pela mesma letra maiúscula, na linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo
teste de Duncan (P ≤ 0,05). Sistema de manejo de N(1)
S1
S2
S3
S4
S5
Híbrido
Padrão
Padrão
Padrão
Monitorado
Padrão
Monitorado
Padrão
Monitorado
Média
Rendimento de grãos, t ha-1
P 32R21
9,7
11,1
12,7
12,5
13,5
13,7
12,2
14,1
12,4 a(2)
Premium
7,2
8,6
9,3
8,3
10,7
10,2
10,6
10,8
9,5 b
Média
8,5 F
9,8 E
11,0 CD
10,4 DE
12,1 A
11,9 AB
11,4 BC
12,5 A
C.V. (%)
Sistema de manejo de N = 8,0; Híbrido = 4,1 mento de grãos de dois híbridos de milho, considerando oito sistemas de manejo de N
S, Eldorado do Sul (RS), 1999/2000 Quadro 6. Rendimento de grãos de dois híbridos de milho, considerando oito sistemas de manejo de N. EEA/UFRGS, Eldorado do Sul (RS), 1999/2000 Quadro 6. Rendimento de grãos de dois híbridos de milho, considerando oito sistem
EEA/UFRGS, Eldorado do Sul (RS), 1999/2000 (1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura, respectivamente de, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5 foram
os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro eram
inferiores ao estabelecido nos respectivos estádios de desenvolvimento. G. ARGENTA et al. As leituras no clorofilômetro correspondentes ao nível ade-
quado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento.(2) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas
pela mesma letra maiúscula, na linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo
teste de Duncan (P ≤ 0,05). (1) Os sistemas de manejo S1 a S5 representam padrões de suprimento de N em cobertura, respectivamente de, 0, 70, 170, 350 e
525 kg ha-1. As doses de N foram aplicadas em quatro épocas: no estádio de três a quatro folhas, foram aplicados 0, 10, 25, 50 e
75 kg ha-1, respectivamente, nos sistemas S1 a S5. O restante do N foi aplicado em três doses iguais, nos estádios de seis a sete
folhas, 10 a 11 folhas e de espigamento do milho. Outros três sistemas equivalentes, respectivamente, aos sistemas S3, S4 e S5 foram
os tratamentos monitorados, em que as doses de N somente foram aplicadas quando as leituras obtidas no clorofilômetro eram
inferiores ao estabelecido nos respectivos estádios de desenvolvimento. As leituras no clorofilômetro correspondentes ao nível ade-
quado de N utilizadas foram de 45,4, 52,1, 55,3 e 58,0, respectivamente, para os estádios de três a quatro folhas, seis a sete folhas,
10 a 11 folhas e de espigamento.(2) Médias seguidas de mesma letra minúscula, na coluna, para comparação de híbridos, e seguidas
pela mesma letra maiúscula, na linha, para comparação de sistemas de manejo de N, não diferem significativamente entre si pelo
teste de Duncan (P ≤ 0,05). R. Bras. Ci. Solo, 27:109-119, 2003 118 G. ARGENTA et al. ARGENTA, G.; SILVA, P.R.F.; MIELNICZUK, J. & BORTOLINI,
C.G. Parâmetros de planta como indicadores do nível de
nitrogênio na cultura do milho. Pesq. Agropec. Bras., 37:519-
527, 2001c. adequado, provavelmente haverá resposta à
aplicação suplementar de N. Quanto aplicar? Isto
dependerá de uma série de fatores que deverão ser
levados em consideração: estádio da cultura, tipo e
quantidade de resíduo da cultura anterior, anos de
semeadura direta, condições climáticas, resposta à
adubação nitrogenada nos anos anteriores, híbrido
utilizado e teto de rendimento de grãos. BINFORD, G.D.; BLACKMER, A.M. & MEESE, B.G. Optimal
concentrations of nitrate in corn stalks at maturity. Agron. J., 84:881-887, 1992. G. ARGENTA et al. DWYER, L.M.; ANDERSON, A.M.; MA, B.L.; STEWART, D.W.;
TOLLENAAR, M. & GREGORICH, E. Quantifying the
nonlinearity in chlorophyll meter response to corn leaf
nitrogen concentration. Can. J. Plant Sci., 75:179-182, 1995. Com relação a este ultimo item, cabe salientar
que o presente método visa à obtenção de alto
potencial de rendimento de grãos. Portanto, para
tetos de rendimentos médios e baixos, é necessário
fazer uma adequação do método proposto. EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA -
EMBRAPA. Centro Nacional de Pesquisa de Solos. Sistema
Brasileiro de Classificação de Solos. Brasília, 1999. 412p. Assim, visualizam-se duas linhas principais de
continuidade desta proposta: integração com alguma
característica de solo, também de rápida determi-
nação, como, por exemplo, a avaliação do teor de
nitrato no solo, e ajuste dos valores correspondentes
ao nível adequado de N para tetos diferenciais de
rendimento de grãos de milho. FRAISSE, C.W. Agricultura de precisão: oportunidades e desafios. Curitiba, Universidade Federal do Paraná, 1998. CD-ROM INSTITUTO DE PESQUISA AGROPECUÁRIA - IPAGRO. Seção
de Ecologia Agrícola. Atlas agroclimático do Estado do Rio
Grande do Sul. Porto Alegre, 1989. 210p. LEMAIRE, G. & GASTAL, F.N. N uptake and distribution in
plant canopies. In: LEMAIRE, G., ed. Diagnosis of the
nitrogen status in crops. Berlin, Springer, 1997. p.1-56. MARQUARD, R.D. & TIPTON, J.L. Relationship between
extractable chlorophyll and an in situ method to estimate
leaf greenness. Hortic. Sci., 22:1327, 1987. CONCLUSÕES MOLL, R.H.; JACKSON, W.A. & MIKKELSEN, R.L. Recurrent
selection for maize grain yield: Dry matter and nitrogen
accumulation and partitioning changes. Crop Sci., 34:874-
881, 1994. 1. O monitoramento do nível de N na planta de
milho pelo teor de clorofila na folha, avaliado pelo
clorofilômetro, evidencia-se como um método
eficiente para separar plantas com deficiência e com
nível adequado deste nutriente nos diferentes
estádios vegetativos de desenvolvimento. MORENO, J.A. Clima do Rio Grande do Sul. Porto Alegre,
Secretaria da Agricultura, 1961. 41p. MUZILLI, O. & OLIVEIRA, E.L. O milho no Paraná. Londrina,
Fundação Instituto Agronômico do Paraná, 1992. p.88-95. (Circular, 29) 2. Para diagnosticar o nível de N na planta de
milho, as leituras no clorofilômetro acima de 45,4,
52,1, 55,3 e 58,0, respectivamente, para os estádios
de três a quatro folhas, seis a sete folhas, 10 a
11 folhas e de espigamento, indicam nível adequado
de N, independentemente do híbrido usado. 2. Para diagnosticar o nível de N na planta de
milho, as leituras no clorofilômetro acima de 45,4,
52,1, 55,3 e 58,0, respectivamente, para os estádios
de três a quatro folhas, seis a sete folhas, 10 a
11 folhas e de espigamento, indicam nível adequado
de N, independentemente do híbrido usado. PLÉNET, D. & CRUZ, P. Maize and sorghum. In: LEMAIRE, G.,
ed.. Diagnosis of the nitrogen status in crops. Berlin,
Springer, 1997. p.93-106. RAJCAN, I. & TOLLENAAR, M. Source:sink ratio and leaf
senescence in maize: II. Nitrogen metabolism during grain
filling. Field Crops Res., 60:255-265, 1999. LITERATURA CITADA RAUN, W.R. & JOHNSON, G.V. Improving nitrogen use efficiency
for cereal production. Agron. J., 91:357-363, 1999. ROBERTS, S. & RHEE, J.K. Critical nutrient concentrations
and DRIS analysis of leaf and grain from high-yielding corn. Comm. Soil Sci. Plant Anal., 24:2679-2687, 1993. ARGENTA, G. Monitoramento do nível de nitrogênio na planta
como indicador da adubação nitrogenada em milho. Porto
Alegre, Universidade Federal do Rio Grande do Sul, 2001. 112p. (Tese de doutorado) SCHRÖDER, J.J.; NEETESON, J.J.; WITHAGEN, J.C.M. & NOIJ,
I.G.A.M. Effects of N application on agronomic and
environmental parameters in silage maize production on
sandy soils. Field Crops Res., 58:55-67, 1998. ARGENTA, G. & SILVA, P.R.F. Adubação nitrogenada em milho
implantado em semeadura direta após aveia preta. Ci. Rural, 29:745-754, 1999. SCHRÖDER, J.J.; NEETESON, J.J.; OENEMA, O. & STRUIK,
P.C. Does the crop or the soil indicate how to save nitrogen
in maize production? Reviewing the state of the art. Field
Crops Res., 66:151-164, 2000. ARGENTA, G.; SILVA, P.R.F. & BORTOLINI, C.G. Teor de
clorofila na folha como indicador do nível de N em cereais. Ci. Rural, 31:715-722, 2001a. ARGENTA, G.; SILVA, P.R.F.; BORTOLINI, C.G.; FORSTHOFER,
E.L. & STRIEDER, M.L. Relação entre teor de clorofila
extraível e leitura do clorofilômetro na folha de milho. R. Bras. Fis. Veg., 13:1101-1106, 2001b. SIMS, J.T.; VASILAS, B.L.; GARTLEY, K.L.; MILLKEN, B. &
GREEN, V. Evaluation of soil and plant nitrogen tests for
maize on manured soils of the Atlhantic coast pain. Agron. J., 87:213-222, 1995. R. Bras. Ci. Solo, 27:109-119, 2003 R. Bras. Ci. Solo, 27:109-119, 2003 ADUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... DUBAÇÃO NITROGENADA EM MILHO PELO MONITORAMENTO DO NÍVEL... 119 SINCLAIR, T.R. & MUCHOW, R.C. Effect of nitrogen supply on
maize yield: I. Modeling physiological responses. Agron. J.,
87:632-641, 1995. VARVEL, G.E.; SCHEPERS, J.S. & FRANCIS, D.D. Ability for
in-season correction of nitrogen deficiency in corn using
chlorophyll meters. Soil Sci. Soc. Am. J., 61:1233-1239, 1997. SMEAL, D. & ZHANG, H. Chlorophyll meter evaluation for
nitrogen management in corn. Comm. Soil Sci. Plant Anal.,
25:1495-1503, 1994. VICTORIA, R.L.; PICCOLO, M.C. & VARGAS, A.A.T. O ciclo do
nitrogênio. In: CARDOSO, E.J.B.N.; TSAI, S.M.; NEVES,
M.C.P., coords. Microbiologia do solo. Campinas, Sociedade
Brasileira de Ciência do Solo, 1992. p.105-120. SOLTANPOUR, P.N.; MALAKOUTI, M.J. & RONAGHI, A. Comparison of DRIS and nutrient sufficiency range for corn. Soil Sci. Soc. Am. J., 59:133-139, 1995. WASKOM, R.M.; WESTFALL, D.G.; SPELLMAN, D.E. &
SOLTANPOUR, P.N. Monitoring nitrogen status of corn
with a portable chlorophyll meter. Comm. Soil Sci. Plant
Anal., 27:545-560, 1996. TEDESCO, M.J.; GIANELLO, C. & BISSANI, C.A. Análise de
solo, plantas e outros materiais. 2. ed. Porto Alegre,
Universidade Federal do Rio Grande do Sul, 1995. 174p. (Boletim Técnico de Solos, 5) YADAVA, U.L. A rapid and nondestrutive method to determine
chlorophyll in intact leaves. Hort. Sci., 21:1449-1450, 1986. R. Bras. Ci. Solo, 27:109-119, 2003
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Effect of lime on the stabilization of an expansive clay soil in Algeria
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Effect of lime on the stabilization of an expansive clay soil in Algeria
Abdelmoumen Aala Eddine DRISS , Khelifa HARICHANE, Mohamed GHRICI
Geomaterials Laboratory, Civil Engineering Department, University Hassiba Benbouali of Chlef, Algeri Received January 10, 2022
Revised March 28, 2022
Accepted April 5, 2022
Published online: November 7, 2022
Keywords
Clay soil
Lime
Plasticity
swell
Unconfined compressive strength
Microstructural analysis Received January 10, 2022
Revised March 28, 2022
Accepted April 5, 2022
Published online: November 7, 2022
Keywords
Clay soil
Lime
Plasticity
swell
Unconfined compressive strength
Microstructural analysis Abstract: This paper presents the influence of lime addition on the geotechnical properties
of an expansive Algerian clayey soil. The studied soil was classified as a fat clay (CH) with high
plasticity according to the unified soil classification system (USCS). 2, 4 and 6% lime was
added to the clay soil in order to studied their effect on the physical and mechanical
properties of the studied soil. The pH variation, consistency limits, compaction, swell,
unconfined compressive strength, mineralogical and microstructural analysis are particularly
investigated. For the purpose of studying the effect of curing time on soil strength, treated
specimens were cured for 1, 7 and 28 days. Tests results indicate that the pH of the studied
soil was significantly increased after their treatment with lime, which indicates the beginning
of chemical reactions between lime and clay soil. After lime treatment the studied soil
become more friable and easier to work with a higher unconfined compressive strength
(UCS) due to the flocculation of the soil structure and the production of new cementation
products such as CSH and CAH. © 2022 The authors. Published by Alwaha Scientific Publishing Services, ASPS. This is an open
access article under the CC BY license. mechanical properties of unstable soils. Among these
stabilization
techniques,
there
are
mechanical,
hydromechanical, thermal and chemical methods. Some of
these methods are very old, such as woodpiles, others are
more recent, such as injection and freezing methods. J. Geomec. Geoeng. 1(1): 1-10 (2023) J. Geomec. Geoeng. 1(1): 1-10 (2023) This work is licensed under a Creative Commons Attribution 4.0. License (CC BY 4.0) http://creativecommons.org/licenses/by/4.0/
JOURNAL OF GEOMECHANICS AND GEOENGINEERING | JGG | ISSN 2716-7992 (PRINT)
Available online at https://asps-journals.com/index.php/jgg
https://doi.org/10.38208/jgg.v1i1.413 1. Introduction Generally, lime
stabilization based on two principal reactions causing
immediate and long-term changes in clay soils. Adding lime
to clay soil immediately increases soil pH due to the
dissociation of calcium hydroxide particles (Vitale et al.,
2017). The short-term reaction based on cation exchange,
when the exchangeable monovalent ions in the clay
particles are replaced by the calcium ions which lead to a
reduction in the thickness of the diffused double layer
(DDL). The ion exchange causes flocculation of clay
particles which improve the workability of the soil by
forming an open soil fabric which decreases its plasticity
and increases its hydraulic conductivity. The relatively high
pH of the pore water facilitates the formation of
pozzolanic reaction bases to produce new cementation
gels (Calcium Silicates Hydrates (CSH) and Calcium
Aluminates Hydrates (CAH)) which harden over time and
lead to a considerable strength gain. The soil is composed of 14% sand, 36% silt and 50% clay, as
shown in the grain size distribution curve shown in Fig.1. Fig. 2 shows the location of the soil in the Casagrande
plasticity chart, it has been classified as a fat clay (CH)
according to USCS (ASTM D2487-06). The mineralogical Table 1 Basic properties of the studied clay soil. Table 1 Basic properties of the studied clay soil. Geotechnical Parameters
Values
Color
Grey
Depth (m)
7 - 17
Natural water content (%)
12 - 14
Specific gravity of soil solids
2.67
Bulk density (g/cm3)
1.17
Passing 80 μm sieve (%)
86.68
Atterberg
Limits
LL (%)
52.64
PL (%)
21.18
PI (%)
31.47
Classification (USCS)
CH
Compaction
PROCTOR
Optimum water content (%)
19.55
Maximum dry density (kN/m3)
16.57
Organics matter content (%)
2.42
Content of calcium carbonate (%)
24.33 ulk density (g/cm )
1.17
assing 80 μm sieve (%)
86.68
Atterberg
imits
LL (%)
52.64
PL (%)
21.18
PI (%)
31.47
lassification (USCS)
CH
ompaction
ROCTOR
Optimum water content (%)
19.55
Maximum dry density (kN/m3)
16.57
Organics matter content (%)
2.42
ontent of calcium carbonate (%)
24.33
Fig. 1 Particle size distribution of the clay soil. Fig. 2 Classification of the studied soil according to the
Casagrande plasticity chart. In this context, an experimental study was carried out to
assess the geotechnical and microscopic properties of lime
stabilized a hight plasticity Algerian clay soil. On the
geotechnical level, pH, Atterberg limits, compaction
characteristics, swell and Unconfined compressive strength
were determined before and after lime treatment. 1. Introduction Clay minerals are very fine particles with very high
electrochemical activity produced by the interaction
between electrically charged particles (Davis, 1955). The
presence of low clay mineral content in natural soils
significantly alters their engineering properties (Holtz and
Kovacs 1981). Clay soils have a large specific surface area
and high ion exchange capacity which produce high
plasticity, high volume change capacity such as swelling
and shrinkage, high compressibility and low bearing
capacity (Davis, 1955). Due to these poor properties, clay
soils are considered to be a big problem for civil engineers. The usual method of stabilizing clay soils is to replace it
with
a
more
resistant
material
that
meets
the
requirements of the project. The very high cost of
performing this method has led researchers to look for
alternative methods. In this context, numerous research
and experimental studies have been carried out on the
chemical stabilization of soils which is based on the use of
chemical and / or cementitious additives to improve
certain geotechnical properties of soils. Extensive studies
have been carried out on soil stabilization using various
additives such as lime (Bell, 1989; Driss et al., 2018, 2019-
a, 2021), cement (Osula, 1996; Ouhadi et al., 2014), fly ash
(Sezer et al., 2006; Mir et al., 2014), natural pozzolana Engineers have observed several problems in the
treatment of clay soils due to their poor geotechnical
properties which do not meet the requirements imposed
for the realization of construction projects. Therefore,
many techniques have been developed by geotechnical
engineers to improve the geotechnical characteristics and Corresponding author. a.driss@univ-chlef.dz Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 2 (Harichane et al., 2018; Driss et al., 2021-b) and slag (Cokca
et al., 2009; Mccarthy et al., 2014). (Harichane et al., 2018; Driss et al., 2021-b) and slag (Cokca
et al., 2009; Mccarthy et al., 2014). laboratory according to American standards. The physico-
mechanical properties of clay soil are presented in Table 1. Lime stabilization of cohesive soils is one of the most
useful chemical methods due to the low cost of lime and
their effectiveness in the field of soil treatment and
improvement (Al- Mukhtar et al., 2012; Driss et al. 2021-
a,b). Lime stabilization of clay soils usually results in
decreased plasticity and volume change, increased particle
size, permeability and soil strength. 1. Introduction The
microstructural and the mineralogical behavior of the
studied soil were investigated with the scanning electron
microscopy (SEM) and the X-Ray Diffraction (XRD). The
results of these study provide a deeper understanding of
the influence of lime on the clayey soils. Fig. 1 Particle size distribution of the clay soil. Fig. 1 Particle size distribution of the clay soil. 2.1 Materials Used In the present study, a gray clay soil was used, it was
extracted at a varying depth between 7 and 17 meters
from a project of resumption of landslide at the Pole
University 2000 Educational places in Mansourah–
Tlemcen. For a better understanding of the behavior of the
soil studied, identification tests were carried out in the Fig. 2 Classification of the studied soil according to the
Casagrande plasticity chart. Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 3 Table 2 Physical and chemical properties of the lime used. Physical and chemical properties
Values
Colour
White
Specific gravity
(g/cm3)
2.24
Bulk density (g/cm3)
0.72
The specific surface -blaine-
(cm2/g)
11663
Particle fineness less than 45 μm
(%)
64.87
Normal consistency -vicat E/L
(%)
69.5
Set time -vicat- (min)
Initial
80
Final
40
Calcium oxide [CaO]
(%)
> 83.3
Magnesium oxide [MgO]
(%)
< 0.5
Iron oxide [Fe2O3]
(%)
< 2
Alumina [Al2O3]
(%)
< 1.5
Silica [SiO2]
(%)
< 2.5
Sulfite [SO3]
(%)
< 0.5
Sodium oxide [Na2O]
(%)
0.4 – 0.5
Carbon dioxide [CO2]
(%)
< 5
Calcite [CaCO3]
(%)
< 10 Table 2 Physical and chemical properties of the lime used. UCS, x-ray diffraction and scanning electron microscopy in
order to justify and relate the results of the experiments to
the mineralogical and microstructural changes resulting
from the treatment of soil. The lime contents used in this
study to treat clay soil were 0, 2, 4 and 6. Table 3 present
the combinations used for the soil treatment. In this study, a pH meter was used for the purpose of
determining the acidity or alkalinity of soil materials
suspended in water. Two pH determination procedures
were used to determine the pH value of untreated and
treated clay soil. For the case of the natural clay soil, the
standard test method for soil pH was used as described in
ASTM D4972 (2001). In order to study the variation of the
pH value and the estimation of the lowest lime content
needed for soil stabilization, the standard test method for
using pH to estimate the soil-lime proportion requirement
for soil stabilization was used in accordance with ASTM
D6276 (1999). This test method is based on the
determination of the lime content favouring the increase
of the pH to a minimum value greater than or equal to
12.4. 2.1 Materials Used According to Eades and Grim (1966), this pH value is
necessary to activate both immediate lime-soil reactions
and long-term pozzolanic reactions. Fig. 3 X-ray diffraction of the studied clay soil. The consistency parameters such as plastic limit (PL), liquid
limit (LL) and plasticity index (PI) for treated and untreated
clay soil were determined according to Atterberg limit
tests (ASTM D4318-17). This method can only be
performed on soil particles smaller than 425 µm (No 40
sieve). The studied soil was firstly dried in the open air
before being thoroughly mixed with 0,2,4 and 6 lime. After
adding distilled water to the mixtures, the paste was left in
an airtight container for about 16 hours before testing. Fig. 3 X-ray diffraction of the studied clay soil. Fig. 3 X-ray diffraction of the studied clay soil. composition of the clay soil was determined by X-ray
diffraction (XRD). As shown in Fig. 3, the XRD analysis
indicated that kaolinite, illite, jadeite and chlorites were
the major clay minerals in untreated soil sediments, other
non-clay minerals were also detected in soil samples,
including quartz and calcite. The standard Proctor compaction test in accordance with
ASTM D698
was used
to
determine compaction
parameters such as optimum water content (OMC) and
maximum dry density (MDD) for the various studied
combinations. The soil-lime mixtures have been carefully
mixed with different water contents and leave for 2 hours
before starting tests, this period is equal to the lime setting
time (time that lime needs to react or fully hydrate with
water). The lime (L) used in this study is a hydrated lime produced
by the company SARL-BSM located in the city of Saïda
(south-west of the Algerian national territory), the
chemical and physical properties of the lime are presented
in Table 2. Table 3 Combinations used for stabilization. Combination
L0
L2
L4
L6
Lime (%)
0
2
4
6
Clay soil (%)
100
98
96
94
Lime (%)
0
2
4
6 Table 3 Combinations used for stabilization. 2.2 Sample Preparation and Test Procedure The main objective of this research is to study the effect of
lime addition on the geotechnical properties of an
expansive clay soil. Several laboratory tests were
performed such as pH, Atterberg limits, compaction, swell, Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 4 One-dimensional free swelling tests were carried out on natural
soil samples before and after stabilization according to method A
mentioned in standard ASTM D4546-03, and under a constant
overpressure of 1 kPa by means of a series of conventional
oedometric devices. First, the sample is prepared at the optimum
water content and the maximum dry density determined
according to the normal Proctor compaction test. The samples
were placed in the oedometers and were flooded with water. The
axial deformation or swelling was then recorded during various
swelling stages (0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 8.0, 15.0 and 30.0 min
and 1, 2, 4, 8, 24, 48 and 72 h) up to a point where axial swelling
has reached an equilibrium value defined as swelling potential or
free swelling percentage. When the soil expansion reaches its
maximum value, the applied vertical load is increased to maintain
the original position. This process is continued until there is no
more swelling (its volume returns to their initial value), The
corresponding stress representing the swelling pressure. One-dimensional free swelling tests were carried out on natural
soil samples before and after stabilization according to method A
mentioned in standard ASTM D4546-03, and under a constant
overpressure of 1 kPa by means of a series of conventional
oedometric devices. First, the sample is prepared at the optimum
water content and the maximum dry density determined
according to the normal Proctor compaction test. The samples
were placed in the oedometers and were flooded with water. The
axial deformation or swelling was then recorded during various
swelling stages (0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 8.0, 15.0 and 30.0 min
and 1, 2, 4, 8, 24, 48 and 72 h) up to a point where axial swelling
has reached an equilibrium value defined as swelling potential or
free swelling percentage. When the soil expansion reaches its
maximum value, the applied vertical load is increased to maintain
the original position. This process is continued until there is no
more swelling (its volume returns to their initial value), The
corresponding stress representing the swelling pressure. Fig. 3.2 Atterberg limits Fig. 5 represent the effect of lime stabilization on the
variation of the Atterberg limits of clay soil. It can be
observed that the addition of lime increases both the
liquid limit (LL) and the plastic limit (PL). But the rate of
increase of LL is lower than that of PL, resulting in a
decrease in the plasticity index (PI). In order to study the effect of lime treatment on the
microstructure and mineralogical behaviour of clay soil,
scanning electron microscopy (SEM) and X-ray diffraction
(XRD) tests were carried out with a MiniFlex 600 type XRD
diffractometer and a JEOL JSM-6060lv Scanning Electron
Microscope. The liquid limit of the clay soil equal to 52.64%, this value
up to 62% after the addition of 2% lime, beyond this
content, the LL has remained almost constant at 4% lime
(62.37%) before dropping slightly at 6%. The plastic limit
increased from 21.18% to 39.41% with the addition of 4%
lime, after this percentage, the plastic limit remains almost 3 Results and Discussion Fig. 5 Influence of lime content on the Atterberg limits 2.2 Sample Preparation and Test Procedure 4 Influence of the addition of lime on the variation of
the pH. Fig. 4 Influence of the addition of lime on the variation of
the pH. produces a highly alkaline environment, due to OH- anions
from the hydration of lime, which leads to slow dissolution
of silica and alumina from the particles of clay. The same
behaviour was observed by Driss et al. 2021-b when
studied the effect of natural pozzolana on microstructural
behavior and hydraulic conductivity of lime‑stabilized
clayey soil Unconfined compressive strength (UCS) tests were carried
out on samples that compacted with their optimum
compaction characteristics in accordance with ASTM
D2166 (2016). Cylindrical specimens with 38 mm diameter
and 80 mm height were compacted by the static
compaction method. The masses of samples were
determined immediately after preparation and then kept
in plastic bags to prevent the moisture change, thus
hardened during the different curing periods such as 1, 7
and 28 days, after curing and before carrying out tests, the
mass and dimensions of samples were recorded. The
strength of specimens was recorded during the UCS test
until the sample fail. 3.1 pH variation Fig. 4 shows the variation in the pH value of the clay soil
studied before and after treatment with different contents
of lime. The studied clay soil is a moderately alkaline soil
with a pH value of 8.3. The clay soil is very reactive to lime. As shown in Fig. 4, a
considerable increase in pH was observed after the
treatment of clay soil with lime. Adding 6% lime to the clay
soil raised its pH from 8.3 to 12.7. This significant increase
in the pH value is explained by the short-term reaction of
lime-clay soil. As known, the addition of lime to clay soils Fig. 5 Influence of lime content on the Atterberg limits Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 5 Fig. 6 Effect of adding lime on soil classification. Fig. 7 Variation of compaction curves. Fig. 7 Variation of compaction curves. Fig. 7 Variation of compaction curves. Fig. 6 Effect of adding lime on soil classification. Fig. 6 Effect of adding lime on soil classification. Fig. 7 Variation of compaction curves. clay soil has a single peak compaction curve with a
maximum dry density (MDD) of 16.57 kN/m3 and an
optimal moisture content (OMC) of 19.55% close to its
plastic limit (21%), these results are consistent with the
model proposed by Gurtug and Sridharan (2004), when the
authors report that there is a good correlation between
OMC and the plastic limit of the cohesive soils (the optimal
moisture content almost equal to 92% of the plastic limit
with a correlation coefficient of 0.95). The compaction
curves of the lime treated clay soil retain their shape
(single peak) with a right pull-down, which means that the
MDD has decreased and the OMC has increased constant. The same behavior has been observed by
Kinuthia et al. (1999). For liquid limit and plastic limit, it
was found that the LL of pure kaolinite increases
considerably after the addition of 3% lime. Above this
content, the LL remained constant even at high
percentages of lime before decreasing slightly at 20%. The
PL also increased significantly with the addition of lime and
remains constant at a lime content varying between 3%
and 14%. The increase in LL and PL after lime treatment
can be explained by the short-term reactions (cation
exchange and agglomeration), which make the soil more
granular and friable. 3.1 pH variation Changes in the structure from
relatively dispersed to flocculated soil particles cause
development of shear strength at the particle level which
increases both of LL and PL. Fig. 8 indicated that adding lime to the clay soil decreases
their MDD and increases the OMC. When the lime content
increased from 0 to 6%, the MDD decreased by 8.6% and
the OMC increased by 16%. As reported by Bell (1989), the
design of lime stabilized soil is proportional to the amount
of lime added (more lime, lower MDD and higher OMC). The results of the consistency limit tests were plotted on
the Casagrande plasticity chart to determine the
classification of the treated soils according to the unified
classification system of soils (USCS) (Fig. 6). As noted, the
studied clay soil is classified as high plasticity clay soil (CH). After the treatment with the different lime contents, their
class was transformed to high plasticity silt class (MH). These changes in soil classification are due to the
flocculation of clay particles based on the cation exchange
between lime and clay particles. The same results were
observed by Harichane et al. (2018). According to the literature, the decrease in dry density can
be explained by the low-density value of lime and the
flocculation of soil particles with immediate formation of
gelatinous compounds (Vitale et al., 2017; Kinuthia et al., Fig. 8 Compaction characteristics of lime-treated clay soil. 3.4 swelling potential and pressure process leads to the formation of attractive forces
between the clay particles, which promotes flocculation of
the particles and the formation of coarse aggregates. Then,
the clay content is reduced which decreases the swelling
potential. After the completion of the cation exchange and
agglomeration, a process called pozzolanic reactions
occurs. This process is strongly dependent on time. During
the pozzolanic reactions, the calcium cations react with
alumina and silica resulting new cementing materials (CSH,
CAH) which promotes solidification of soil caused a
considerable reducing in swelling potential and swelling
pressure. Schanz and Elsawy (2017) found the same
variation in volume change when they investigated the
effect of 5% and 10% lime addition on the free swelling of
expansive clay soil. Fig. 9 Free swell as a function of time for treated and
untreated clay soil. 3.3 Compaction Compaction tests were carried out to determine the effect
of lime on the compaction characteristics (optimum
moisture content and maximum dry density) of the treated
soil. The soil compaction curves before and after treatment,
with 2, 4 and 6% lime are represented in Fig. 7. The studied Fig. 8 Compaction characteristics of lime-treated clay soil. Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 6 (a)
(b)
Fig. 10 Variation of swelling potential and swelling
pressure as a function of lime content. (a)
(b) (a) 1999). The increase in OMC may be due to the increase in
the water holding capacity to supply the amount of water
necessary for pozzolanic reactions produced between clay
particles and the added lime. 3.4 swelling potential and pressure The swell-time curves for natural soils and samples
stabilized with various percentages of lime cured for 1 and
28 days, are shown in Fig. 9. It can be clearly seen from Fig. 9 that the addition of lime to the clayey soil accelerates the
swelling procedure, especially during the early stages of
this phenomenon (up to about 120 min). on the other
hand, lower swelling potential was recorded for almost all
samples stabilized with lime compared to natural soil. Simply put, it can be concluded that it takes less time for
lime-treated samples to approach their maximum swelling
potential. Fig. 10 shows the variation in swelling potential and
swelling pressure of soil samples mixed with lime content
varying between 2% and 6%. It is observed that the
untreated clay sample reaches the maximum swelling
potential (10%). The use of 2% lime in the expansive clay
leads to a significant decrease in swelling potential and
pressure. Fig. 10 Variation of swelling potential and swelling
pressure as a function of lime content. The effect of lime on the swelling behavior of the clayey
soil can be explained by a series of chemical reactions that
take place in the presence of water between the lime and
the clay minerals. These chemical reactions can be divided
into three broad categories, namely cation exchange,
flocculation-agglomeration
and
pozzolanic
reaction. During the cation exchange process, the cations common
to the clay surface, notably sodium (Na+) and potassium
(K+) are replaced by more charged cations, especially
calcium (Ca2+) and in some cases magnesium (Mg2+). This process leads to the formation of attractive forces
between the clay particles, which promotes flocculation of
the particles and the formation of coarse aggregates. Then,
the clay content is reduced which decreases the swelling
potential. After the completion of the cation exchange and
agglomeration, a process called pozzolanic reactions
occurs. This process is strongly dependent on time. During
the pozzolanic reactions, the calcium cations react with
alumina and silica resulting new cementing materials (CSH,
CAH) which promotes solidification of soil caused a
considerable reducing in swelling potential and swelling
pressure. Schanz and Elsawy (2017) found the same
variation in volume change when they investigated the
effect of 5% and 10% lime addition on the free swelling of
expansive clay soil. 3.5 Unconfined Compressive Strength Fig. 11 shows the effect of the addition of lime on the
stress-strain relationships of the studied soil. The first type
of curves, which has an asymptotic appearance without
peak, represents the curves of the clayey soil alone. The
second type represents the curves of soil treated with
lime, containing peaks expressing their maximum stress Fig. 9 Free swell as a function of time for treated and
untreated clay soil. Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 7 Fig. 12 Variation of the unconfined compressive strength
of lime treated clay soil at different curing times 1, 7 and
28 days. Fig. 12 Variation of the unconfined compressive strength
of lime treated clay soil at different curing times 1, 7 and
28 days. (a)
(b) (a)
(b)
(c)
Fig. 11 Stress-strain curves of clay soil treated with
different lime contents and different hardening times
(a) 1 day, (b) 7 days, (c) 28 days. (a) (b) Fig. 12 Variation of the unconfined compressive strength
of lime treated clay soil at different curing times 1, 7 and
28 days. (c) stress gain, when the UCS of the studied soil increased
from 354 kPa to 572 kPa when treated with 6% lime after 1
curing day. A further increase was produced with the
curing time, the UCS of the 6% treated soil increased by
13% and 47% after 7 and 8 days of cure compared to 1
day. These improvement in the compressive strength
based on two principal factors; short-term reactions such
as cation exchange and the flocculation or agglomeration
of soil particles which make the soil more granular and
brittle (Al-Mukhtar et al., 2012). On the other hand, there
is a significant increase in the UCS after 28 days of
hardening compared to the untreated soil and the treated
soils at short curing times (1day and 7 days), this increase
is attributed to the pozzolanic reaction of lime with the
particles of clay soil which produced new cementing
compounds, thus binding the soil particles together. (c) 4. Conclusions (a) (a)
(c)
(e) An experimental investigation was conducted to study the
effect of lime addition on pH variation, Atterberg limits,
compaction
parameters,
swell
and
unconfined
compressive strength of an expansive clay soil. In light of
the test results, the following conclusions were drawn: (d) (c) The increase in lime content for stabilized clay soil changes
its moderately alkaline pH to a high alkaline pH value, this
increase in pH is a sign of the start of chemical reactions
between the soil and lime. Addition of lime to clay soil increased both LL and PL,
causing a considerable decrease in PI due to the change in
the structure of soil particles, which changes the behaviour
of the soil from dispersed to flocculated. (e) (f) (f) (e) The design of the lime stabilized soil corresponds to the
added lime content; more lime led to a decrease in MDD
and an increase in the OMC. Fig. 13 SEM microphotographs of samples of clay before
and after treated with lime. (a) and (b) clay soil, (c) and
(d) lime treated soil at 1day of hardening, (e) and (f) lime
treated soil at 28 days of hardening. A significant reduction in swelling parameters was noted
for the lime-treated samples with further improvement
upon increasing curing time. The unconfined compressive strength of the treated clay
soil increased with the increase in both lime content and
hardening time, this behaviour can be explained by the
considerable modification of the soil structure caused by
the short- and long-term reactions. Fig. 14 X-ray diffraction patterns of soil treated and
untreated with lime at 1 day and 28 days of hardening. Fig. 14 X-ray diffraction patterns of soil treated and
untreated with lime at 1 day and 28 days of hardening. After 28 curing days (Fig. 13 (e, f)), the flocculated clay
particles were covered by a white hydrated gel, which
means the formation of new cementitious compounds
such as C-S-H and C-A-H (Fig. 14). The short- and long-term
reactions have caused the considerable change in the
behaviour of the clay soil when the soil changes from
ductile to brittle with higher compressive strength. The mineralogical and microstructural analyses confirmed
that the addition of lime to the clay soil produced an
important change in their microstructure when the soil
become more friable. 3.6 Mineralogical and microstructural analysis Fig. 13 and 14 represent the effect of lime treatment on
the
microstructure
(SEM)
and
the
mineralogical
composition (XRD) of the studied soil at 1 and 28 curing
days. As shown in Fig. 13 (a, b) the compacted clay soil has
an evident massive close packing structure with a
dispersed
arrangement. The
same
microstructural
structure was observed by Rosone et al. (2018) when
treated a similar class of clay soil (CH) with lime. The
microstructure
of
the
studied
soil
was
changed
immediately after the addition of lime. As reported in Fig. 13 (c, d) in the short term, a modification of the clay
particles arrangement was observed with formation of
larger aggregates compared to not treated soil (particles
soil flocculation) without any evidence of new hydrated
phases (Fig. 14). Fig. 11 Stress-strain curves of clay soil treated with
different lime contents and different hardening times
(a) 1 day, (b) 7 days, (c) 28 days. value followed by a reduction in stress (residual stress)
until the sample failure. So, it can be noted that the clay
soil behaves as a ductile material with a plastic
deformation in their natural state. In the other side, when
treated the clay soil with lime their behaviour was
transforming from ductile to brittle. The same behaviour
was observed by Yıldız and Soğancı (2012) and Kavak and
Baykal (2012). Fig. 12 represents the variation in the UCS of the studied
clay soil treated with 2, 4 and 6% lime at 1, 7 and 28 curing
days. Adding lime to the clay soil produced considerable Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 8 (a)
(b)
(c)
(d)
(e)
(f)
Fig. 13 SEM microphotographs of samples of clay before
and after treated with lime. (a) and (b) clay soil, (c) and
(d) lime treated soil at 1day of hardening, (e) and (f) lime
treated soil at 28 days of hardening. (b) Disclosures Fig. 14 X-ray diffraction patterns of soil treated and
untreated with lime at 1 day and 28 days of hardening. Fig. 14 X-ray diffraction patterns of soil treated and
untreated with lime at 1 day and 28 days of hardening. Free
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to
this
article
is
sponsored
by
SARL ALPHA CRISTO INDUSTRIAL. Free
Access
to
this
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sponsored
by
SARL ALPHA CRISTO INDUSTRIAL. After 28 curing days (Fig. 13 (e, f)), the flocculated clay
particles were covered by a white hydrated gel, which
means the formation of new cementitious compounds
such as C-S-H and C-A-H (Fig. 14). The short- and long-term
reactions have caused the considerable change in the
behaviour of the clay soil when the soil changes from
ductile to brittle with higher compressive strength. After 28 curing days (Fig. 13 (e, f)), the flocculated clay
particles were covered by a white hydrated gel, which
means the formation of new cementitious compounds
such as C-S-H and C-A-H (Fig. 14). The short- and long-term
reactions have caused the considerable change in the
behaviour of the clay soil when the soil changes from
ductile to brittle with higher compressive strength. 4. Conclusions In the same time, showed that at 28
curing days new cementitious phases was produced
(pozzolanic
reactions)
which
induced
significant
improvements in the engineering properties of the clayey
soil such as their workability, compaction and compressive
strength. References Al-Mukhtar, M., Lasledj, A., & Alcover, J.-F. (2010). Behaviour and
mineralogy changes in lime-treated expansive soil at 20°C. Applied Clay Science, 50(2), 191–198. Driss, A. A. E., Harichane, K., Ghrici, M., & Gadouri, H. (2021-a). Assessing the effect of moulding water content on the
behaviour of lime-stabilised an expansive soil. Geomechanics
and Geoengineering, 1-13. Al-Mukhtar,
M.,
Khattab,
S.,
&
Alcover,
J.-F. (2012). Microstructure and geotechnical properties of lime-treated
expansive clayey soil. Engineering Geology, 139-140, 17–27. Driss, A. A. E., Harichane, K., & Ghrici, M. (2021-b). Effect of
natural pozzolana on microstructural behavior and hydraulic
conductivity of lime-stabilized clayey soil. Innovative
Infrastructure Solutions, 6(4), 1-17. ASTM D2166 / D2166M-16. (2016). Standard Test Method for
Unconfined Compressive Strength of Cohesive Soil, ASTM
International, West Conshohocken, PA. ASTM D2487 (2017), Standard Practice for Classification of Soils
for Engineering Purposes (Unified Soil Classification System),
ASTM International, West Conshohocken, PA. Eades JL, Grim RE (1966) A quick test to determine lime
requirements for lime stabilization. Highway Research Record
n°139. ASTM D4318-05. (2005). Standard Test Methods for Liquid Limit,
Plastic Limit, and Plasticity Index of Soils, ASTM International,
West Conshohocken, PA. Garzón, E., Cano, M., O`Kelly, B. C., & Sánchez-Soto, P. J. (2016). Effect of lime on stabilization of phyllite clays. Applied Clay
Science, 123, 329–334. ASTM D4546-03 (2003), Standard Test Methods for One-
Dimensional Swell or Settlement Potential of Cohesive Soils,
ASTM International, West Conshohocken, PA, www.astm.org Gurtug, Y., & Sridharan, A. (2004). Compaction Behaviour and
Prediction of its Characteristics of Fine Grained Soils with
Particular Reference to Compaction Energy. Soils and
Foundations,44(5),27–36. ASTM D4972-01 (2001) Standard Test Method for pH of Soils,
ASTM International, West Conshohocken. www.astm.org Harichane, K., Ghrici, M., & Kenai, S. (2018). Stabilization of
Algerian Clayey Soils with Natural Pozzolana and Lime. Periodica Polytechnica Civil Engineering. 62(1), 1-10. ASTM D6276-19 (2019) Standard Test Method for Using pH to
Estimate the Soil-Lime Proportion Requirement for Soil
Stabilization. ASTM International, West Conshohocken. www.astm.org Harichane, K., Ghrici, M., Kenai, S., & Grine, K. (2011). Use of
Natural Pozzolana and Lime for Stabilization of Cohesive Soils. Geotechnical and Geological Engineering, 29(5), 759–769. ASTM D698-12e2. (2012). Standard Test Methods for Laboratory
Compaction Characteristics of Soil Using Standard Effort (12
400 ft-lbf/ft3 (600 kN-m/m3)), ASTM International, West
Conshohocken, PA. Hossain, K. M. A., Lachemi, M., & Easa, S. (2007). Stabilized soils
for construction applications incorporating natural resources
of Papua new Guinea. Resources, Conservation and Recycling,
51(4), 711–731. Acknowledgments This research was sponsored by the General Directorate
for Scientific Research and Technological Development
(DGRSDT) of the Algerian Minister of Higher Education and
Scientific Research. The authors thank the Head of
Engineering
Civil
Department
and
the
Laboratory Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 9 argileux. In 8th International Symposium on construction in
seismic zone (SICZS 2018), Chlef on (Vol. 10). engineers of Belhadj Bouchaib University of Aïn-
Temouchent for their contribution to the laboratory tests
and the carrying out of this research. Driss, A. A. E., Harichane, K., Ghrici, M., Gadouri, H., & GOUFI, A. (2019) Stabilization of expansive clay soil based on initial
consumption of lime. In 1st International Congress on
Advances in Geotechnical Engineering and Construction
Management ICAGECM’19 (p. 285). References Attoh-Okine, N. O. (1995). Lime treatment of laterite soils and
gravels — revisited. Construction and Building Materials, 9(5),
283–287. Jha, A. K., & Sivapullaiah, P. V. (2015). Mechanism of
improvement in the strength and volume change behavior of
lime stabilized soil. Engineering Geology, 198, 53–64. Bell FG (1989) Lime stabilization of clay soils. Bulletin of the
International Association of Engineering Geology 39:67-74. Kavak, A., & Baykal, G. (2012). Long-term behavior of lime-
stabilized kaolinite clay. Environmental Earth Sciences, 66(7),
1943–1955. Cokca, E., Yazici, V., & Ozaydin, V. (2008). Stabilization of
Expansive Clays Using Granulated Blast Furnace Slag (GBFS)
and GBFS-Cement. Geotechnical and Geological Engineering,
27(4), 489–499. Kinuthia, J., Wild, S., & Jones, G. (1999). Effects of monovalent
and divalent metal sulphates on consistency and compaction
of lime-stabilised kaolinite. Applied Clay Science, 14: 27–45. Davis LE (1955) Electrochemical Properties of Clays. Clays and
Clay Minerals, 1(1): 47–53. Manasseh, J., & Olufemi, A. I. (2008). Effect of lime on some
geotechnical properties of Igumale shale. Electronic Journal
of Geotechnical Engineering, 13(5), 1-9. Driss, A. A. E., Harichane, K., & Ghrici, M. (2018) Effet de la chaux
sur la stabilisation des proprietes geotechniques d’un sol Driss et al. / J. Geomec. Geoeng. 1(1): 1-10 (2023) 10 McCarthy, M. J., Csetenyi, L. J., Sachdeva, A., & Dhir, R. K. (2014). Engineering and durability properties of fly ash treated lime-
stabilised sulphate-bearing soils. Engineering Geology, 174,
139–148. Schanz, T., & Elsawy, M. B. (2017). Stabilisation of highly swelling
clay using lime–sand mixtures. Proceedings of the Institution
of Civil Engineers-Ground Improvement, 170(4), 218-230. Sezer, A., İnan, G., Yılmaz, H. R., & Ramyar, K. (2006). Utilization
of a very high lime fly ash for improvement of Izmir clay. Building and Environment, 41(2), 150–155. Mir, B. A., & Sridharan, A. (2013). Physical and Compaction
Behaviour of Clay Soil–Fly Ash Mixtures. Geotechnical and
Geological Engineering, 31(4), 1059–1072. Stoltz, G., Cuisinier, O., & Masrouri, F. (2012). Multi-scale analysis
of the swelling and shrinkage of a lime-treated expansive
clayey soil. Applied Clay Science, 61, 44–51. Osula, D. O. A. (1996). A comparative evaluation of cement and
lime modification of laterite. Engineering Geology, 42(1), 71–
81. Vitale, E., Deneele, D., Paris, M., & Russo, G. (2017). Multi-scale
analysis and time evolution of pozzolanic activity of lime
treated clays. Applied Clay Science, 141, 36–45. Ouhadi, V. R., Yong, R. N., Amiri, M., & Ouhadi, M. H. (2014). Pozzolanic consolidation of stabilized soft clays. References Applied Clay
Science, 95, 111–118. Yıldız, M., & Soğancı, A. S. (2012). Effect of freezing and thawing
on strength and permeability of lime-stabilized clays. Scientia
Iranica, 19(4), 1013–1017. Rao, S. M., & Shivananda, P. (2005). Compressibility behaviour of
lime-stabilized clay. Geotechnical and Geological Engineering,
23(3), 309–319. Yong, R., & Ouhadi, V. (2007). Experimental study on instability of
bases on natural and lime/cement-stabilized clayey soils. Applied Clay Science, 35(3-4), 238– -249. Rosone, M., Celauro, C., & Ferrari, A. (2018). Microstructure and
shear strength evolution of a lime-treated clay for use in road
construction. International Journal of Pavement Engineering,
1–12.
|
https://openalex.org/W3123644889
|
https://ojs.unud.ac.id/index.php/jbn/article/download/66821/37793
|
Indonesian
| null |
Mutasi Isocitrate Dehydrogenase 1 dan 2 pada Glioma
|
JBN (Jurnal Bedah Nasional)
| 2,020
|
cc-by
| 2,443
|
ABSTRAK Klasifikasi tumor sistem saraf pusat WHO terakhir didasarkan atas histogenesis tumor dan karakteristik
molekuler genetik. Perubahan klasifikasi yang paling signifikan terjadi pada tumor glioma difus yang
paling signifikan, yaitu katagori tumor secara garis besar berdasarkan status mutasi isocitrate
dehydrogenase (IDH) 1 dan 2. Tulisan ini akan membahas tentang IDH1 dan IDH baik normal maupun
mutan, hubungan mutasi IDH1 dan IDH2 dengan prognosis pasien, serta bagaimana penentuan status
mutasi IDH1 dan IDH2 dalam diagnosis glioma. Kata kunci: glioma, IDH, isocitrate dehydrogenase, mutasi. Mutasi Isocitrate Dehydrogenase 1 dan 2 pada Glioma Mutasi Isocitrate Dehydrogenase 1 dan 2 pada Glioma ABSTRACT The latest WHO central nervous system classification is based on tumor histogenesis and genetic
molecular characteristics. The most significant classification change is in diffuse gliomas, which is
tumor categorization mainly based on isocitrate dehydrogenase (IDH) 1 and 2 mutation status. This
review will be described about normal and mutant IDH1 and IDH2, correlation between IDH mutation
status with prognosis, and how to determine IDH1 and IDH2 mutation status in diagnosis of gliomas. Keywords: glioma, IDH, isocitrate dehydrogenase, mutation. Ni Putu Sriwidyani* Departemen Patologi Anatomi Fakultas Kedokteran Universitas Udayana. *Penulis korespondensi: sriwidyani@unud.ac.id. 26 | JBN (Jurnal Bedah Nasional) IDH1 DAN IDH2 132 (R132) sedangkan mutasi pada IDH2 pada
asam amino arginine 172 (R172).8 132 (R132) sedangkan mutasi pada IDH2 pada
asam amino arginine 172 (R172).8 132 (R132) sedangkan mutasi pada IDH2 pada
asam amino arginine 172 (R172).8 IDH pada manusia terdapat dalam tiga
isoform, yaitu IDH1, IDH2, dan IDH3. IDH1
(terdapat di sitoplasma dan peroksisom) dan
IDH2 (terdapat di matriks mitokondria)
merupakan nicotinamide adenine dinucleotide
phosphate
(NADP+)-dependent. Enzim
homodimer ini mengkatalisir reaksi redox
yang mengkonversi isocitrate menjadi α-
ketoglutarate (α-KG) saat mereduksi NDP
menjadi NADPH dan melepaskan CO2. Enzim IDH1 dan IDH2 berperan pada
metabolism karbohidrat, asam lemak, dan
glutamate, serta berkontribusi dalam menjaga
status redox seluler normal.3 Pada tumorigenesis glioma, diduga mutasi
gen yang menyandi IDH1 dan IDH2 akan
menghasilkan protein mutan (2-HG) yang
dapat menginhibisi diferensisi sel punca
glioma, menimbulkan upregulasi vascular
endothelial growth factor (VEGF) yang akan
memicu pembentukan lingkungan mikro
tumor, dan meningkatkan level hypoxia-
inducible factor-1α (HIF-1α).9 Mutasi IDH
tampaknya merupakan proses onkogenik awal
pada gliomagenesis, dimana mutasi-mutasi
lanjutan merupakan proses penting yang
berkontribusi terhadap pembentukan tumor. Mutasi pada gen IDH1 dan IDH2 yang
menyandi protein IDH1 dan IDH2 akan
menghasilkan
protein
mutan
yang
menginhibisi aktifitas enzim normal. Di
samping itu, enzim IDH mutan memiliki
aktifitas neomorfik yang akan mengkonversi
α-KG menjadi 2-HG.3 PENDAHULUAN IDH merupakan suatu enzim yang berperan
pada siklus tricarboxylic acid (TCA) yang
penting dalam metabolisme energi. IDH
mutan akan menghasilkan metabolit 2-
hydroxyglutaric acid (2-HG) yang tampaknya
berperan pada gliomagenesis pada kasus
glioma difus dengan IDH mutan.3,4,5 Mutasi
IDH dijumpai pada lebih dari 70% kasus
glioma difus grade II/III dan glioblastoma
sekunder.6,7 Glioma merupakan tumor sistem saraf
pusat (SSP) tersering, terutama pada dewasa. Pada klasifikasi tumor SSP tahun 2016,
Klasifikasi tumor SSP mengintegrasikan
gambaran morfologi dan molekuler. Di
samping berdasarkan gambaran histologik,
tumor
glioma
difus
pada
dewasa
diklasifikasikan berdasarkan status mutasi gen
isocitrate dehydrogenase (IDH), p53 dan
alpha
thalassemia/mental
retardation
syndrome x-linked (ATRX), serta adanya ko-
delesi 1p/19q.1 Pemahaman tentang genetik
molekuler
pada
glioma
menjadi
dasar
klasifikasi tumor, serta penentuan prognosis
dan pilihan terapi pasien glioma.2 Tulisan ini akan membahas tentang IDH1
dan IDH2 normal dan mutan, serta implikasi
pemeriksaan status IDH1 dan IDH2 pada
diagnosis terintegrasi pada glioma. 26 | JBN (Jurnal Bedah Nasional) Ni Putu Sriwidyani JBN (Jurnal Bedah Nasional) STATUS
IDH
DAN
DIAGNOSIS
GLIOMA
MENURUT
KLASIFIKASI
WHO TAHUN 2016 Klasifikasi tumor saraf pusat WHO sejak
edisi pertama sampai keempat didasarkan atas
konsep histogenesis dengan mengevaluasi
morfologi tumor untuk menilai dari sel mana
tumor berasal. Dengan banyaknya penelitian
molekuler yang meningkatkan pengetahuan
tentang perubahan genetik yang mendasari
tumorigenesis tumor SSP, maka klasifikasi
tumor SSP terbaru (WHO edisi keempat
(update)
tahun
2016)
mengintegrasikan
gambaran histologik dan molekuler dalam
klasifikasi tumor SSP. MUTASI
IDH1
DAN
IDH2
PADA
GLIOMA IDH1
dan
IDH2
terlibat
dalam
tumorigenesis
beberapa
jenis
keganasan
seperti glioma, leukemia myeloid akut,
limfoma, kolangiokarsinoma intrahepatik, dan
kondrosarkoma.2 Pada glioma difus, mutasi
IDH ini tampaknya berperan pada proses awal
tumorigenesis pada sebagian besar kasus
glioma grade II dan III, serta sebagian kasus
glioblastoma sekunder. Pada
klasifikasi
tumor
SSP
edisi
sebelumnya, glioma difus pada dewasa
diklasifikasikan menjadi tumor astrositik,
oligodendroglial, dan oligoastrositik. Pada
edisi tahun 2016, di samping berdasarkan
gambaran histologik tumor, klasifikasi juga
didasarkan
atas
berdasarkan
perubahan
genetik yaitu status mutasi IDH, p53 dan
ATRX,
serta
adanya
ko-delesi
1p/19q
(Gambar 1).1 Penelitian oleh Parsons dkk. (2008)
merupakan yang pertama kali megidentifikasi
adanya mutasi IDH1 pada 12% sampel kasus
glioblastoma
yang
diteliti. Penelitian-
penelitian selanjutnya menunjukkan adanya
mutasi IDH1 dan IDH2 pada 70-90% kasus
glioma grade II-III dan glioblastoma sekunder. Mutasi IDH1 yang dijumpai adalah missense
mutation pada satu asam amino pada arginine 27 27 Volume 5 ● Number 1 ● Januari 2021 Mutasi Isocitrate Dehydrogenase Gambar 1. Algoritme klasifikasi glioma difus berdasarkan gambaran histologik dan genetik.1
Ko-delesi 1p/19q
& parameter
genetik lain
Status IDH
Glioblastoma
IDH mutant
IDH wild type
Glioblastoma, IDH mutant
Glioblastoma, IDH wild type
Astrositoma difus, IDH mutant
Oligodendroglioma, IDH
mutant & kodelesi 1p/19q
Astrositoma difus, IDH wild type
Oligodendroglioma, IDH wild type
Oligoastrositoma
IDH mutant
IDH wild type
ATRX loss
Mutasi
TP53
Kodelesi
1p/19q Oligoastrositoma Glioblastoma IDH wild type IDH mutant Ko-delesi 1p/19q
& parameter
genetik lain Glioblastoma, IDH mutant ATRX loss
Mutasi Kodelesi
1p/19q Glioblastoma, IDH wild type Astrositoma difus, IDH mutant Oligodendroglioma, IDH
mutant & kodelesi 1p/19q Astrositoma difus, IDH wild type
Oligodendroglioma, IDH wild type Gambar 1. Algoritme klasifikasi glioma difus berdasarkan gambaran histologik dan genetik.1 Gambar 2. Anaplastic astrocytoma, WHO 2016. (a) Inti sel berbentuk oval relatif uniform dengan latar
belakang mikrokistik. (b) Pada pembesaran tinggi, beberapa inti telanjang tanpa prosesus sitoplasma,
sementara sebagian lainnya menunjukkan prosesus fibriler, inti sel menunjukkan iregularitas dan
hiperkromasia. Gambaran ini sesuai untuk anaplastic astrocytoma. (c) Imunohistokimia IDH1 R132H
negatif. (d) p53 positif difus. (e) Imunoreaktivitas ATRX hilang pada sel tumor tetapi intak pada sel
endotel. (f) Karena p53 positif dan ATRX negatif, kasus cenderung dengan mutasi IDH. Sanger
sequencing menunjukkan adanya mutasi IDH1 R132L.11 Gambar 2. Anaplastic astrocytoma, WHO 2016. (a) Inti sel berbentuk oval relatif uniform dengan latar
belakang mikrokistik. (b) Pada pembesaran tinggi, beberapa inti telanjang tanpa prosesus sitoplasma,
sementara sebagian lainnya menunjukkan prosesus fibriler, inti sel menunjukkan iregularitas dan
hiperkromasia. MUTASI
IDH1
DAN
IDH2
PADA
GLIOMA Kesulitan
lainnya adalah dalam membedakan glioma
dengan lesi non neoplastik dan membedakan
glioma dengan lesi neoplastik SSP primer
lainnya. Spesimen dari tumor SSP sering pula
hanya berupa fragmen jaringan kecil yang
menimbulkan kesulitan tersendiri dalam
diagnosis. Deteksi adanya mutasi IDH1 dan IDH2
dapat dilakukan dengan beberapa metode. Studi awal tentang mutasi IDH pada glioma
blok parafin, dan metode ini paling sering
dilakukan pada klinis untuk menentukan
klasifikasi tumor. Tahapan diagnosis kasus glioma difus yang
saat
ini
dilakukan
di
banyak
senter
laboratorium
patologi
yaitu:
1)Evaluasi
histologik untuk mengkonfirmasi asal tumor;
2)Pemeriksaan
imunohistokimia
IDH1
R132H; 3)Pemeriksaan imunohistokimia p53
dan ATRX; 4)FISH untuk memeriksa ko-
delesi 1p/19q; 5)Jika imunohistokimia IDH1
R132H menunjukkan hasil negatif, maka
dimintakan pemeriksaan sequencing mutasi
IDH1/IDH2; 6)Jika histologik menunjukkan
glioblastoma
(grade
IV),
dimintakan
pemeriksaan
metilasi
O-6-
methylguaninetransferase
(MGMT).2
(Gambar 2)
Saat ini terapi target yang mentarget mutasi
IDH2 pada leukemia myeloid akut telah
disetujui di Amerika, namun terapi target pada
glioma dengan mutasi IDH1 dan IDH2 masih
dalam tahap penelitian. Meski mekanisme
yang mendasari muatasi IDH berperan dalam
biologi tumor dan terapi target masih dalam
tahap penelitian, kepentingan klinik deteksi
mutasi IDH pada diagnosis glioma ini akan
menentukan klasifikasi, dan stratifikasi risiko
pasien. Pasien glioma grade II dan III dengan
mutasi IDH memiliki angka survival yang
lebih baik dibandingkan pasien tanpa mutasi
IDH.11
SIMPULAN
Mutasi
IDH1
dan
IDH2
merupakan Pada
WHO
2016,
evaluasi
sediaan
konvensional
H&E
tetap
merupakan
stratifikasi awal. Setelah menentukan kategori
mayor (seperti infiltrating glioma, neuronal
tumor, atau embryonal tumor) berdasarkan
histologik, kemudian dilanjutkan dengan hasil
pemeriksaan molekuler. Terdapat status “not
otherwise specified (NOS)” untuk kasus
dimana pemeriksaan genetik tidak tersedia,
pemeriksaan genetik tidak menunjukkan
gambaran alterasi genetik yang sesuai dengan
temuan
histologik,
atau
jika
terdapat
keterbatasan evaluasi karena sampel jaringan
yang
insufisien
atau
terdapat
artefak
jaringan.10 blok parafin, dan metode ini paling sering
dilakukan pada klinis untuk menentukan
klasifikasi tumor. Tahapan diagnosis kasus glioma difus yang
saat
ini
dilakukan
di
banyak
senter
laboratorium
patologi
yaitu:
1)Evaluasi
histologik untuk mengkonfirmasi asal tumor;
2)Pemeriksaan
imunohistokimia
IDH1
R132H; 3)Pemeriksaan imunohistokimia p53
dan ATRX; 4)FISH untuk memeriksa ko-
delesi 1p/19q; 5)Jika imunohistokimia IDH1
R132H menunjukkan hasil negatif, maka
dimintakan pemeriksaan sequencing mutasi
IDH1/IDH2; 6)Jika histologik menunjukkan
glioblastoma
(grade
IV),
dimintakan
pemeriksaan
metilasi
O-6-
methylguaninetransferase
(MGMT).2
(Gambar 2) Integrasi pemeriksaan molekuler di atas
pada pemeriksaan histologik konvensional
memberikan jalan keluar pada masalah yang
cukup sering dijumpai pada diagnosis glioma
berdasarkan evaluasi morfologi. y g
(Gambar 2) Saat ini terapi target yang mentarget mutasi
IDH2 pada leukemia myeloid akut telah
disetujui di Amerika, namun terapi target pada
glioma dengan mutasi IDH1 dan IDH2 masih
dalam tahap penelitian. Meski mekanisme
yang mendasari muatasi IDH berperan dalam
biologi tumor dan terapi target masih dalam
tahap penelitian, kepentingan klinik deteksi
mutasi IDH pada diagnosis glioma ini akan
menentukan klasifikasi, dan stratifikasi risiko
pasien. Pasien glioma grade II dan III dengan
mutasi IDH memiliki angka survival yang
lebih baik dibandingkan pasien tanpa mutasi
IDH.11 Deteksi adanya mutasi IDH1 dan IDH2
dapat dilakukan dengan beberapa metode. Studi awal tentang mutasi IDH pada glioma
menggunakan direct sequencing. Meskipun
sequencing merupakan gold standard untuk
deteksi mutasi IDH, telah dikembangkan alat
deteksi yang lebih cepat dan lebih mudah
dilakukan. Pengembangan
antibodi
monoklonal
terhadap
IDH1
R132H
memungkinkan pemeriksaan adanya mutasi
IDH yang paling sering terjadi pada glioma
yang dapat dilakukan pada jaringan dalam MUTASI
IDH1
DAN
IDH2
PADA
GLIOMA Evaluasi
sediaan H&E pada kasus glioma sering sulit
membedakan antara astrositoma grade II
dengan
grade
III
karena
kesulitan
membedakan selularitas, derajat atipia, dan
tidak adanya cut-off jumlah mitosis. Kesulitan
lainnya adalah dalam membedakan glioma
dengan lesi non neoplastik dan membedakan
glioma dengan lesi neoplastik SSP primer
lainnya. Spesimen dari tumor SSP sering pula
hanya berupa fragmen jaringan kecil yang
menimbulkan kesulitan tersendiri dalam
diagnosis. MUTASI
IDH1
DAN
IDH2
PADA
GLIOMA Gambaran ini sesuai untuk anaplastic astrocytoma. (c) Imunohistokimia IDH1 R132H
negatif. (d) p53 positif difus. (e) Imunoreaktivitas ATRX hilang pada sel tumor tetapi intak pada sel
endotel. (f) Karena p53 positif dan ATRX negatif, kasus cenderung dengan mutasi IDH. Sanger
sequencing menunjukkan adanya mutasi IDH1 R132L.11 28 Ni Putu Sriwidyani JBN (Jurnal Bedah Nasional) Pada
WHO
2016,
evaluasi
sediaan
konvensional
H&E
tetap
merupakan
stratifikasi awal. Setelah menentukan kategori
mayor (seperti infiltrating glioma, neuronal
tumor, atau embryonal tumor) berdasarkan
histologik, kemudian dilanjutkan dengan hasil
pemeriksaan molekuler. Terdapat status “not
otherwise specified (NOS)” untuk kasus
dimana pemeriksaan genetik tidak tersedia,
pemeriksaan genetik tidak menunjukkan
gambaran alterasi genetik yang sesuai dengan
temuan
histologik,
atau
jika
terdapat
keterbatasan evaluasi karena sampel jaringan
yang
insufisien
atau
terdapat
artefak
jaringan.10
Integrasi pemeriksaan molekuler di atas
pada pemeriksaan histologik konvensional
memberikan jalan keluar pada masalah yang
cukup sering dijumpai pada diagnosis glioma
berdasarkan evaluasi morfologi. Evaluasi
sediaan H&E pada kasus glioma sering sulit
membedakan antara astrositoma grade II
dengan
grade
III
karena
kesulitan
membedakan selularitas, derajat atipia, dan
tidak adanya cut-off jumlah mitosis. Kesulitan
lainnya adalah dalam membedakan glioma
dengan lesi non neoplastik dan membedakan
glioma dengan lesi neoplastik SSP primer
lainnya. Spesimen dari tumor SSP sering pula
hanya berupa fragmen jaringan kecil yang
menimbulkan kesulitan tersendiri dalam
diagnosis. Deteksi adanya mutasi IDH1 dan IDH2
dapat dilakukan dengan beberapa metode. Studi awal tentang mutasi IDH pada glioma
menggunakan direct sequencing
Meskipun
b
d
k
s
l
h
2
R
d
d
R
d
I
g
p
m
(
I
d
g
d
y
b
t
m
m
p
m
l
I
S
p Pada
WHO
2016,
evaluasi
sediaan
konvensional
H&E
tetap
merupakan
stratifikasi awal. Setelah menentukan kategori
mayor (seperti infiltrating glioma, neuronal
tumor, atau embryonal tumor) berdasarkan
histologik, kemudian dilanjutkan dengan hasil
pemeriksaan molekuler. Terdapat status “not
otherwise specified (NOS)” untuk kasus
dimana pemeriksaan genetik tidak tersedia,
pemeriksaan genetik tidak menunjukkan
gambaran alterasi genetik yang sesuai dengan
temuan
histologik,
atau
jika
terdapat
keterbatasan evaluasi karena sampel jaringan
yang
insufisien
atau
terdapat
artefak
jaringan.10
Integrasi pemeriksaan molekuler di atas
pada pemeriksaan histologik konvensional
memberikan jalan keluar pada masalah yang
cukup sering dijumpai pada diagnosis glioma
berdasarkan evaluasi morfologi. Evaluasi
sediaan H&E pada kasus glioma sering sulit
membedakan antara astrositoma grade II
dengan
grade
III
karena
kesulitan
membedakan selularitas, derajat atipia, dan
tidak adanya cut-off jumlah mitosis. PERNYATAAN Penulis tidak memiliki konflik kepentingan
dengan pihak lain dalam penulisan ini. Penulis tidak memiliki konflik kepentingan
dengan pihak lain dalam penulisan ini. 7. Monga V, Jones K, Chang S. Clinical
Relevance Of Molecular Markers In
Gliomas. Rev Médica Clínica Las Condes. 2017;28:343-51. SIMPULAN Mutasi IDH1 dan IDH2 merupakan
peristiwa onkogenik awal pada glioma derajat
rendah. Mutasi ini ditemukan pada sebagian
besar kasus glioma difus pada dewasa. Klasifikasi tumor SSP WHO tahun 2016
mengintegrasikan gambaran histologik dan
genetik molekuler termasuk pada glioma. Integrasi ini penting dalam menentukan
prognosis dan rencana terapi pada pasien. Ke
depan, diperlukan banyak penelitian tentang 29 Volume 5 ● Number 1 ● Januari 2021 Mutasi Isocitrate Dehydrogenase FM, dkk. Cancer-associated mutation and
beyond:
The
emerging
biology
of
isocitrate
dehydrogenases
in
human
disease. Sci Adv.2019;5:eaaw4543. mekanisme mutasi IDH dalam gliomagenesis
dan biologi tumor, serta penelitian tentang
terapi target yang mentarget IDH mutan
sehingga meningkatkan peluang memperbaiki
outcome pasien glioma. 6. Turkalp Z, Karamchandani J, Das S. IDH
mutation in glioma: New insights and
promises for the future. JAMA Neurol. 2014;71:1319-25. 6. DAFTAR PUSTAKA 1. Louis DN, Perry A, Reifenberger G, dkk. The 2016 World Health Organization
Classification of Tumors of the Central
Nervous System: a summary. Acta
Neuropathol. 2016;131:803-20. 1. Louis DN, Perry A, Reifenberger G, dkk. The 2016 World Health Organization
Classification of Tumors of the Central
Nervous System: a summary. Acta
Neuropathol. 2016;131:803-20. 8. Yan H, Parsons DW, Jin G, dkk. IDH1 and
IDH2 mutations in gliomas. N Engl J Med. 2009;360:765-73. 9. Huang J, Yu J, Tu L, dkk. Isocitrate
dehydrogenase mutations in glioma: From
basic
discovery
to
therapeutics
development. Front Oncol. 2019;9:506. 2. Lee
SC. Diffuse
gliomas
for
nonneuropathologists: The new integrated
molecular diagnostics. Arch Pathol Lab
Med. 2018;142:804-14. 10. Olar A, Wani KM, Alfaro-Munoz KD,
dkk. IDH mutation status and role of WHO
grade and mitotic index in overall survival
in grade II–III diffuse gliomas. Acta
Neuropathol.2015; 129:585-596 3. Cairns RA, Mak TW. Oncogenic isocitrate
dehydrogenase mutations: Mechanisms,
models, and clinical opportunities. Cancer
Discov. 2013;3:730-41. 11. Komori T. The 2016 WHO classification
of tumours of the central nervous system:
The major points of revision. Neurol Med
Chir (Tokyo). 2017;57:301-11. 4. Maus A, Peters GJ. Glutamate and α-
ketoglutarate: key players in glioma
metabolism. Amino Acids. 2017;49:21-32. 5. Tommasini-Ghelfi S, Murnan K, Kouri 5. Tommasini-Ghelfi S, Murnan K, Kouri 30 30
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Multifunctional layered magnetic composites
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Address: Address:
1Department of Chemistry, Physical Chemistry, University of
Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany, 2Jülich
Centre for Neutron Science JCNS-MLZ, Outstation at MLZ,
Forschungszentrum Jülich, Lichtenbergstraße 1, 85748 Garching,
Germany, 3Theoretical Chemistry, University of Erlangen-Nürnberg,
Nägelsbachstraße 25, 91052 Erlangen, Germany, 4Physical
Chemistry II, University of Bayreuth, Universitätsstraße 30, 95447
Bayreuth, Germany, 5Laboratory for Neutron Scattering, Paul
Scherrer Institute, 5232 Villigen PSI, Switzerland, 6Technische
Universität München, Forschungs-Neutronenquelle Heinz
Maier-Leibnitz (FRM II), 85748 Garching, Germany and 7Department
of Biomaterials, Max Planck Institute of Colloids & Interfaces Science
Park Golm, 14424 Potsdam, Germany Email:
Helmut Cölfen* - helmut.coelfen@uni-konstanz.de Helmut Cölfen* - helmut.coelfen@uni-konstanz.de * Corresponding author Keywords: bio-inspired mineralization; biomineralization; chitin; ferrogel; hybrid
materials; magnetite; nacre Maria Siglreitmeier1, Baohu Wu1,2, Tina Kollmann3, Martin Neubauer4, Gergely Nagy5,
Dietmar Schwahn6, Vitaliy Pipich2, Damien Faivre7, Dirk Zahn3, Andreas Fery4
and Helmut Cölfen*1 Full Research Paper
Open Access
Address:
1Department of Chemistry, Physical Chemistry, University of
Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany, 2Jülich
Centre for Neutron Science JCNS-MLZ, Outstation at MLZ,
Forschungszentrum Jülich, Lichtenbergstraße 1, 85748 Garching,
Germany, 3Theoretical Chemistry, University of Erlangen-Nürnberg,
Nägelsbachstraße 25, 91052 Erlangen, Germany, 4Physical
Chemistry II, University of Bayreuth, Universitätsstraße 30, 95447
Bayreuth, Germany, 5Laboratory for Neutron Scattering, Paul
Scherrer Institute, 5232 Villigen PSI, Switzerland, 6Technische
Universität München, Forschungs-Neutronenquelle Heinz
Maier-Leibnitz (FRM II), 85748 Garching, Germany and 7Department
of Biomaterials, Max Planck Institute of Colloids & Interfaces Science
Park Golm, 14424 Potsdam, Germany
Email:
Helmut Cölfen* - helmut.coelfen@uni-konstanz.de
* Corresponding author
Keywords:
bio-inspired mineralization; biomineralization; chitin; ferrogel; hybrid
materials; magnetite; nacre
Beilstein J. Nanotechnol. 2015, 6, 134–148. doi:10.3762/bjnano.6.13
Received: 02 June 2014
Accepted: 09 December 2014
Published: 12 January 2015
This article is part of the Thematic Series "Towards multifunctional
inorganic materials: biopolymeric templates". Guest Editors: C. Steinem and J. Bill
© 2015 Siglreitmeier et al; licensee Beilstein-Institut. License and terms: see end of document. Full Research Paper
Open Access
Address:
1Department of Chemistry, Physical Chemistry, University of
Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany, 2Jülich
Centre for Neutron Science JCNS-MLZ, Outstation at MLZ,
Forschungszentrum Jülich, Lichtenbergstraße 1, 85748 Garching,
Germany, 3Theoretical Chemistry, University of Erlangen-Nürnberg,
Nägelsbachstraße 25, 91052 Erlangen, Germany, 4Physical
Chemistry II, University of Bayreuth, Universitätsstraße 30, 95447
Bayreuth, Germany, 5Laboratory for Neutron Scattering, Paul
Scherrer Institute, 5232 Villigen PSI, Switzerland, 6Technische
Universität München, Forschungs-Neutronenquelle Heinz
Maier-Leibnitz (FRM II), 85748 Garching, Germany and 7Department
of Biomaterials, Max Planck Institute of Colloids & Interfaces Science
Park Golm, 14424 Potsdam, Germany
Email:
Helmut Cölfen* - helmut.coelfen@uni-konstanz.de
* Corresponding author
Keywords:
bio-inspired mineralization; biomineralization; chitin; ferrogel; hybrid
materials; magnetite; nacre
Beilstein J. Nanotechnol. 2015, 6, 134–148. doi:10.3762/bjnano.6.13
Received: 02 June 2014
Accepted: 09 December 2014
Published: 12 January 2015
This article is part of the Thematic Series "Towards multifunctional
inorganic materials: biopolymeric templates". Guest Editors: C. Steinem and J. Bill
© 2015 Siglreitmeier et al; licensee Beilstein-Institut. License and terms: see end of document. Multifunctional layered magnetic composites Maria Siglreitmeier1, Baohu Wu1,2, Tina Kollmann3, Martin Neubauer4, Gergely Nagy5,
Dietmar Schwahn6, Vitaliy Pipich2, Damien Faivre7, Dirk Zahn3, Andreas Fery4
and Helmut Cölfen*1 Introduction Recent work underlines the importance of amor-
phous precursor phases [17] and also nonclassical crystalliza-
tion mechanisms in biomineralization [18,19]. Biominerals, which are organic–inorganic hybrids and highly
sophisticated materials with optimal assimilated properties,
have evolved in nature. The mechanisms of biomineral forma-
tion are still far from being understood and there is currently
large research activity from groups of different expertise. Most
biominerals are hierarchically structured, which consequently
adds favorable physical properties such as hardness and frac-
ture resistance to the material. An intriguing and much investi-
gated material is nacre which is the inner protecting layer of
some marine sea shells. It is well-known for its beautiful irides-
cence but also for the outstanding mechanical properties. Nacre
has a layered structure of aragonite platelets and an organic
matrix mainly consisting of β-chitin covered with proteins [1]. This hybrid structure makes nacre 3000 times more fracture
resistant as compared to aragonite which makes up ca. 95 wt %
of this structure [2]. The reason for this is that crack propaga-
tion is hindered by the soft chitin layers that get disrupted
before the crack can propagate further. In addition, the platelets
are glued to the organic matrix by elastic proteins that also have
sacrificial physical bonds [3]. Another amazing biomineral are
chiton teeth, which are actually the hardest known biomineral
[4]. Chitons scratch algae from rocks, which requires wear-
resistant teeth. The animal maintains this ability by synthe-
sizing rows of teeth and each time, a tooth is worn out, the next
tooth in the row will be used. A reason for the mechanical wear
resistance of the teeth is the presence of different iron oxide
mineral phases incorporated into a protein–polysaccharide
matrix. Especially, magnetite nanoparticles that are present in
large amounts (ca. 70 wt %) at the tooth cap, covering the
cutting surface, are responsible for the outstanding mechanical
performance [5]. In this manuscript we report a synthesis method to combine the
favorable properties of two biominerals in one and the same
material and, thus, to create a multifunctional hybrid material. We claim that this bioinspired material could find potential
application in various fields. In general, it could be very
interesting for the field of abrasive and fracture resistant ma-
terials that are found in hard coatings or in the field of construc-
tion. Introduction We used the organic nacre matrix of the shell Haliotis
laevigata, which is insoluble in acetic acid, as a confined reac-
tion environment. Within this organic matrix we infiltrated
gelatin to mimic the silk gel precursor inside the chitin nacre
scaffold [3]. Inside this organic gelatin matrix we synthesize
magnetite nanoparticles to form a highly mineralized
organic–inorganic hybrid body. The resulting material should
mimic the fracture resistance of nacre and the hardness and
abrasion resistance of the chiton teeth. Abstract A fabrication method of a multifunctional hybrid material is achieved by using the insoluble organic nacre matrix of the Haliotis
laevigata shell infiltrated with gelatin as a confined reaction environment. Inside this organic scaffold magnetite nanoparticles
(MNPs) are synthesized. The amount of MNPs can be controlled through the synthesis protocol therefore mineral loadings starting
from 15 wt % up to 65 wt % can be realized. The demineralized organic nacre matrix is characterized by small-angle and very-
small-angle neutron scattering (SANS and VSANS) showing an unchanged organic matrix structure after demineralization
compared to the original mineralized nacre reference. Light microscopy and confocal laser scanning microscopy studies of stained
samples show the presence of insoluble proteins at the chitin surface but not between the chitin layers. Successful and homoge-
neous gelatin infiltration in between the chitin layers can be shown. The hybrid material is characterized by TEM and shows a
layered structure filled with MNPs with a size of around 10 nm. Magnetic analysis of the material demonstrates superparamagnetic
behavior as characteristic for the particle size. Simulation studies show the potential of collagen and chitin to act as nucleators,
where there is a slight preference of chitin over collagen as a nucleator for magnetite. Colloidal-probe AFM measurements demon-
strate that introduction of a ferrogel into the chitin matrix leads to a certain increase in the stiffness of the composite material. 134
134 Beilstein J. Nanotechnol. 2015, 6, 134–148. Introduction fundamental knowledge of the underlying mechanisms as well
as theoretical explanations were, so far, only provided for rare
examples. One of the reasons is that many of the biomineraliza-
tion mechanisms are still not fully understood due to their
complexity. Recent work underlines the importance of amor-
phous precursor phases [17] and also nonclassical crystalliza-
tion mechanisms in biomineralization [18,19]. Biominerals, which are organic–inorganic hybrids and highly
sophisticated materials with optimal assimilated properties,
have evolved in nature. The mechanisms of biomineral forma-
tion are still far from being understood and there is currently
large research activity from groups of different expertise. Most
biominerals are hierarchically structured, which consequently
adds favorable physical properties such as hardness and frac-
ture resistance to the material. An intriguing and much investi-
gated material is nacre which is the inner protecting layer of
some marine sea shells. It is well-known for its beautiful irides-
cence but also for the outstanding mechanical properties. Nacre
has a layered structure of aragonite platelets and an organic
matrix mainly consisting of β-chitin covered with proteins [1]. This hybrid structure makes nacre 3000 times more fracture
resistant as compared to aragonite which makes up ca. 95 wt %
of this structure [2]. The reason for this is that crack propaga-
tion is hindered by the soft chitin layers that get disrupted
before the crack can propagate further. In addition, the platelets
are glued to the organic matrix by elastic proteins that also have
sacrificial physical bonds [3]. Another amazing biomineral are
chiton teeth, which are actually the hardest known biomineral
[4]. Chitons scratch algae from rocks, which requires wear-
resistant teeth. The animal maintains this ability by synthe-
sizing rows of teeth and each time, a tooth is worn out, the next
tooth in the row will be used. A reason for the mechanical wear
resistance of the teeth is the presence of different iron oxide
mineral phases incorporated into a protein–polysaccharide
matrix. Especially, magnetite nanoparticles that are present in
large amounts (ca. 70 wt %) at the tooth cap, covering the
cutting surface, are responsible for the outstanding mechanical
performance [5]. fundamental knowledge of the underlying mechanisms as well
as theoretical explanations were, so far, only provided for rare
examples. One of the reasons is that many of the biomineraliza-
tion mechanisms are still not fully understood due to their
complexity. Results and Discussion
Synthetic concept It is the aim to synthesize a material of larger dimensions by
developing a multifunctional biomimetic composite structure,
which combines properties of two biominerals in one and the
same material, namely nacre and chiton teeth. To reach this goal
we follow the key synthesis principles presented in Figure 1. The starting material is an original demineralized nacre matrix
that is infiltrated by a thermo reversible gelatin solution mimic- There are many approaches to produce an organic–inorganic
hybrid material inspired by the structure of nacre [6-16]. But the Figure 1: Magnetite formation inside a gelatin gel matrix (grey) that is placed inside the chitin scaffold of demineralized nacre (dark grey lines). Panel
a) symbolizes the stage of mineralization after one reaction cycle, panel b) represents repeated mineralization cycles as demonstrated by the
progress of mineralization shown by the increase of the magnetite nanoparticle number. At zero time, only the gel matrix is present. Figure 1: Magnetite formation inside a gelatin gel matrix (grey) that is placed inside the chitin scaffold of demineralized nacre (dark grey lines). Panel
a) symbolizes the stage of mineralization after one reaction cycle, panel b) represents repeated mineralization cycles as demonstrated by the
progress of mineralization shown by the increase of the magnetite nanoparticle number. At zero time, only the gel matrix is present. 135 Beilstein J. Nanotechnol. 2015, 6, 134–148. the nacre and its organic matrix over a wide range of length
scales from about 1 nm to 1 μm. The data in Figure 2 show
several distinct Q-regimes that are described well by the solid
line representing the best fit of the data using Beaucage’s
expression [30] and a correlation model [31] (see Supporting
Information File 1). For nacre, scattering from the aragonite
mesocrystals is dominant in the Q-regime less than 0.02 nm−1
and is represented by a Q−2 power law with an amplitude of
P2 = 1.8 cm−1·nm−2. This exponent implies a platelet-like struc-
tural characteristic with a plate diameter larger than 2 μm as
evaluated from the radius of gyration, Rg, assuming the form
factor of a thin plate-like shape [32]. Above Q* 0.063 nm−1
the power law transforms into Q−3 and above 0.4 nm−1 to a Q−4
Porod behavior that yields an average size of the nanograins of
about 10 nm as estimated from D ≡ 2π/Q*. Nacre organic matrix – SANS Nacre, an inorganic and organic composite natural material, is
typically found as the inner shells of mollusks, and is referred to
as mother-of-pearl. Its structure is a layered arrangement of
pseudo-hexagonally shaped aragonite mesocrystals with a diam-
eter of around 10–15 μm and a thickness of about 500 nm [3]
(every platelet consists of polygonal CaCO3 nanograins with a
size of 10–45 nm [20]). The aragonite mesocrystals are inter-
spaced by an organic matrix which was identified as a β-chitin
[21-23] core surrounded by protein layers that play an impor-
tant role in the formation process of nacre [24-26]. The inor-
ganic mesocrystals are connected by mineral bridges with a
width ranging from 36–54 nm in between the neighboring
lamellae. The mineral bridges represent the continuation of
mineral growth along the vertical direction of the lamellar
mesocrystals from a preceding layer of platelets [27,28]. The
fraction of the organic matrix in nacre is only about 5 wt %, it
plays an important role in the spatial control of mineralization,
hierarchical structure and toughness enhancement [23,29]. Different techniques have been used to resolve the chemical and
structural composition of the organic matrix. Small angle
neutron scattering (SANS) is a non-destructive method to study
the nacreous organic matrix without potential changes to the
matrix, which might derive from the usage of staining media or
dehydration. For comparison studies, the structure of the orig-
inal nacre matrix (Haliotis laevigata) was analyzed as well. Figure 2 represents very-small (VSANS) and small (SANS)
angle neutron scattering profiles of nacre (top) and its organic
matrix (bottom) measured at two diffractometers for very small
(VSANS) and conventional small angular scattering (SANS) in,
respectively, Q-ranges from 10−3 to 2·10−2 nm–1 and from 10−2
to 3.5 nm−1. The absolute value of the scattering vector Q is
related to the scattering angle θ and neutron wavelength λ
according to Q = (4π/λ)·sin(θ/2). The neutron beam is parallel to
the c-axis of the nacre or the organic matrix of the nacre (i.e.,
perpendicular to the sample surface, see Supporting Informa-
tion File 1). Thus, nearly no information about the thickness of
the lamellar platelets is found in the scattering curves. Results and Discussion
Synthetic concept The diameter of the
nanograins is around 11 nm as evaluated from Rg, assuming the
form factor of a spherical shape, which is consistent with data
reported in literature [29]. The scattering, which follows the
Q−2 power law between 3·10−2 and 0.2 nm−1 shows the pres-
ence of a shoulder which might correspond to the mineral
bridges with an average diameter that is estimated to be
D ≈ 80 nm from Rg ≈ 28.9 nm. king a gel precursor inside the chitin nacre scaffold. Within this
gelatin matrix we synthesize magnetite nanoparticles to form a
highly mineralized organic–inorganic hybrid body. This hybrid
structure resembles the nacre aragonite platelets in size and
shape. We repeated this reaction cycle up to eight times in order
to enhance and thereby control the content of magnetite NPs
inside the hybrid material. Systematic studies showed that after
eight reaction cycles the upper limit of mineral load is reached
and further repetition does not lead to an increase in the mineral
content. Nacre organic matrix – SANS In summary, we can conclude
that nacre is completely demineralized by our experimental
procedure, which was also confirmed by TGA measurements,
and that the structure of the demineralized nacre organic matrix
has not significantly changed compared with the original nacre. arrangement of gelatin in between the insoluble organic nacre
matrix layers a Coomassie stain is used. The light microscopy
image in Figure 3a shows an embedded and thin cut section of
demineralized nacre stained with Coomassie blue. The investi-
gations clearly display a blue stain of the layered insoluble
nacre structure as a result of a positive interaction of the insol-
uble proteins with Coomassie blue, whereas the space in
between the layers does not show any significant stain. The
same observation can be made by fluorescence confocal laser
scanning microscopy (Figure 3b) for which the thin cuts have
been stained with rhodamine B ITC. Also in these studies no
staining of proteins in between the layers could be observed. Therefore we conclude that the insoluble matrix proteins are
dominantly located directly at the β-chitin matrix and are not
present in between the layers. Figure 3c and Figure 3d show an
embedded sample of insoluble nacre matrix infiltrated with
gelatin by a vacuum infiltration process. Staining of this sample
illustrates not only blue stained chitin layers and insoluble
matrix proteins but also colored areas in between the layers,
indicating a filling of the matrix with gelatin. The interaction
and positive stain of gelatin and Coomassie blue have been
tested successfully in reference experiments (see Figure S3,
Supporting Information File 1). For a better visualization of the
stained areas in between the layers the light blue stained gelatin
(see Supporting Information File 1) has been processed digi-
tally. This means the green RGB channel of the images was
exchanged by the red one to be able to better distinguish
between the different matrix parts. As a result the stained
gelatin parts appear purple in the image which makes it easier to
differentiate between the blue chitin layers and the filling in
between the layers. The purple area next to the matrix in
Figure 3d represents excess of gelatin on the sample surface. These studies reveal that the chitin–gelatin composite can be
used as a template for the mineralization of magnetite and there-
fore act as a building block for the formation of a multifunc-
tional composite material. Nacre organic matrix – Light microscopy and
fluorescence microscopy Original nacre (Haliotis laevigata) used for materials synthesis
was analyzed by light microscopy and confocal fluorescence
laser scanning microscopy (FCLSM) as can be seen in Figure 3. A freshly broken cross section of original nacre was analyzed
by SEM (see below in Figure 4c) and clearly reveals the layered
structure of aragonite tablets. The insoluble organic matrix can
be seen in Figure 3 and in the transmission electron microscopy
(TEM) image given below in Figure 5d. The embedded cross
section of the demineralized chitin matrix shows that the matrix
remains stable after demineralization and does not stick
together. These results are in agreement with our findings from
SANS and VSANS experiments, therefore we conclude that the
demineralized nacre matrix can be used as a template for the
synthesis of the composite material, which is in agreement with
earlier work on nacre retrosynthesis [13]. The distance between
the layers is around 250–500 nm (see below in Figure 5d),
which is in part lower than that of the natural archetype
(500 nm) due to a partial collapse of the demineralized matrix
during preparation and handling. Figure 5d also illustrates
vertical connections between the layers, these thin walls are the
so-called “intertabular matrix” which has a stabilizing function
[33]. The interruptions in the layers correspond to pores of
around 50–70 nm thickness and act as mineral transport bridges
during the formation of natural nacre, as also confirmed by
SANS and VSANS experiments. In order to determine the Nacre organic matrix – SANS One key step for the formation of
such a multifunctional hybrid material is the homogeneous infil-
tration of gelatin as the organic scaffold for mineralization
inside the insoluble nacre matrix. Nacre organic matrix – SANS These
measurements enable the determination of the hierarchical
structures along the vertical direction of the lamellar platelets of Figure 2: SANS macroscopic cross-section dΣ/dΩ versus scattering
vector Q for a 1 mm thick piece of nacre in air and a demineralized
nacre matrix in D2O (T = 20 °C). The neutron beam is parallel to the
nacre/nacre organic matrix c-axis (perpendicular to the sample
surface). At low Q (<0.02 nm−1) VSANS data are also presented after
rescaling. The solid line represents a fit of the Beaucage equation [30]
and correlation length model (Q > 0.03 nm−1) [31] (see Supporting
Information File 1). Figure 2: SANS macroscopic cross-section dΣ/dΩ versus scattering
vector Q for a 1 mm thick piece of nacre in air and a demineralized
nacre matrix in D2O (T = 20 °C). The neutron beam is parallel to the
nacre/nacre organic matrix c-axis (perpendicular to the sample
surface). At low Q (<0.02 nm−1) VSANS data are also presented after
rescaling. The solid line represents a fit of the Beaucage equation [30]
and correlation length model (Q > 0.03 nm−1) [31] (see Supporting
Information File 1). 136 Beilstein J. Nanotechnol. 2015, 6, 134–148. The scattering profile of the nacre organic matrix (Figure 2,
bottom) indicates the same platelet-like structure as for nacre as
it shows the same power laws, however with an amplitude of
P2 = 0.13 cm−1·nm−2, which is one order of magnitude smaller. This means that the demineralization has no significant influ-
ence on the original structure of the organic matrix. Above
Q = 0.03 nm−1 a radius of gyration Rg of about 24.3 nm is
determined, which might correspond to the mineral bridges. The
diameters of the cross section of the bridges were estimated to
be roughly D ≈ 68 nm from Rg ≈ 24.3 nm. This result is consis-
tent with our TEM results. The size of the mineral bridge is
much larger than the typical size of the gelatin molecule as
determined from the correlation length ξ ≈ 15.9 ± 0.5 nm with
SAXS (see Supporting Information File 1). This indicates that
the molecular diffusion of gelatin into the organic chitin matrix
through holes in the chitin layers, which originates from the
former mineral bridges, is possible. Above Q = 0.5 nm−1 scat-
tering from around 0.8 nm large particles appear, representing
the scattering of the chitin chain. General synthesis protocol and TEM/SEM
studies The synthesis of the multifunctional inorganic hybrid material is
based on an already established three step protocol [34]. In the
first step, the gelatin hydrogel is infiltrated into the demineral-
ized nacre matrix through a vacuum infiltration process [35],
in the second step this chitin–gelatin composite is introduced
into a solution of ferrous (FeCl2 0.1 M) and ferric ions
(FeCl3 0.2 M) in a molar ratio of 1:2. After complete diffusion
of the ions inside the hydrogel template magnetite is precipi-
tated in the third step by introducing the template in a base 137 Beilstein J. Nanotechnol. 2015, 6, 134–148. Figure 3: Light microscopy image of thin cuts of embedded and Coomassie stained samples. a) Demineralized nacre matrix, b) confocal laser scan-
ning microscopy of embedded demineralized nacre matrix stained with rhodamine B ITC, c) and d) demineralized nacre matrix (blue) with infiltrated
gelatin (purple). Figure 3: Light microscopy image of thin cuts of embedded and Coomassie stained samples. a) Demineralized nacre matrix, b) confocal laser scan-
ning microscopy of embedded demineralized nacre matrix stained with rhodamine B ITC, c) and d) demineralized nacre matrix (blue) with infiltrated
gelatin (purple). (NaOH 0.1 M). The magnetite nanoparticles are synthesized
through a so-called co-precipitation method following the reac-
tion: (NaOH 0.1 M). The magnetite nanoparticles are synthesized
through a so-called co-precipitation method following the reac-
tion: loading of the composite material with iron oxide nanoparticles
varies from 15–65 wt % depending on the number of reaction
cycles (see Figure S3, Supporting Information File 1). Scan-
ning electron microscopy (SEM) examinations of the dried
hybrid materials indicate a dense layered hierarchical structure
(see Figure 4a), which is similar to natural nacre. The distribu-
tion of magnetite nanoparticles inside the hybrid material was
determined with electron dispersive X-ray spectroscopy (EDX)
(Figure 4d). The mapping of the elements shows that Fe and C
are homogeneously distributed throughout the material surface
whereas there is less C detected at the freshly broken cross
section of the material. It can be clearly seen that the spaces in
between the layers mainly give signals for Fe. With the
performed studies we could not observe a mineral gradient
throughout the matrix arising from the synthesis of the magnetic
nanoparticles produced by a diffusion approach. Therefore we
claim that after full completion of the synthesis the particles are
equally present over the whole matrix. General synthesis protocol and TEM/SEM
studies (1) (1) (1) This procedure can be repeated several times in order to obtain
the desired degree of mineralization. We already reported a
similar synthesis protocol for gelatin-based magnetic hydrogels
[34] and now transfer these synthesis principles into the insol-
uble organic nacre matrix. The amount of magnetite nanoparticles formed inside the
synthesized hybrid material was determined by thermogravi-
metric measurements. The initial and final degradation tempera-
tures have been determined from the thermogram curves. The 138 Beilstein J. Nanotechnol. 2015, 6, 134–148. Figure 4: SEM micrographs of a) and b) fracture surfaces of artificial nacre and c) fracture surface of original nacre Haliotis laevigata. d) EDX
mapping analysis of artificial nacre fracture surface. Figure 4: SEM micrographs of a) and b) fracture surfaces of artificial nacre and c) fracture surface of original nacre Haliotis laevigata. d) EDX
mapping analysis of artificial nacre fracture surface. of magnetite through an amorphous or ferrihydrite precursor
stage. However, the transformation of amorphous iron oxide
species into magnetite was observed before and is also likely to
happen in this synthesis set-up [36]. Reference experiments of
the composite material without gelatin infiltration (Figure 5c)
and repetition of four reaction cycles only show the presence of
nanoparticles adsorbed on the chitin surface but not in between
the layers. This material seems closer to the demineralized
nacre matrix (Figure 5d) than to a multilayered composite ma-
terial. Furthermore, the distance in between the layers for
samples containing gelatin seems less collapsed than for
samples without gelatin which results in a material closer in
structure to that one of original nacre. Electron diffraction data
taken from different areas in between the layers show the pres-
ence of polycrystalline nanoparticles with no preferred orienta-
tion (see Figure S5, Supporting Information File 1). The iron
oxides magnetite and maghemite show very similar diffraction
patterns and d-spacings, therefore it is not possible to differen-
tiate these mineral phases with the used techniques. In summary In order to confirm this observation and to obtain information
about the mineral nature in between the chitin sheets, TEM
studies of embedded and microtome cut samples have been
conducted (Figure 5). We note the presence of iron oxide
nanoparticles homogeneously distributed in between the layers
after one (Figure 5a) and four reaction cycles (Figure 5b). General synthesis protocol and TEM/SEM
studies Moreover, it can also be seen that the number of particles after
one reaction cycle is significantly lower than after four reaction
cycles, which is in agreement with TGA studies of the hybrid
materials. The studies show that the particles are in the size
range of 10 ± 5 nm and do exist at the chitin surface as well as
in between the chitin layers due to the presence of the carrying
media gelatin. It is also worth to mention that besides the 10 nm
sized particles also smaller particles in the size range of around
3 nm can be detected. Electron diffraction studies of these small
particles show their amorphous nature, which leads to the
conclusion that under the chosen synthesis conditions amor-
phous material or poorly crystallized ferrihydrite could be
present. In this study we could not recognize a direct formation 139 Beilstein J. Nanotechnol. 2015, 6, 134–148. Figure 5: TEM micrographs of a) artificial nacre after one reaction cycle and b) after four reaction cycles, c) reference chitin–magnetite composite
sample without gelatin, and d) completely demineralized matrix. Figure 5: TEM micrographs of a) artificial nacre after one reaction cycle and b) after four reaction cycles, c) reference chitin–magnetite composite
sample without gelatin, and d) completely demineralized matrix. chitin–gelatin composite (top) and as a reference in a gel matrix
(bottom). The structure of the ferrogel (the hybrid material
without chitin) was investigated for comparison. The
magnetite–gelatin–chitin sample shows a power law of Q−1 in
the low-Q regime (<0.01 nm–1), which is approximately valid
for linear structures and thereby indicates rod-like particles or
chains of particles of about Rg = 0.58 μm. At larger
Q (>0.1 nm−1) scattering is determined from individual
magnetite nanoparticles of Rg 7.9 nm showing a Q−3 power
law indicating a mass fractal structure (a structure containing
branching and crosslinking to form a 3D network). The diam-
eter D of the magnetite particles can be estimated to be
D ≈ 20 nm (Rg = D/2.58) assuming a spherical shape. The scat-
tering of magnetite in the gelatin matrix (ferrogel) qualitatively
looks the same. Particles (or an assembly of particles) of about these observations demonstrate that it is possible to success-
fully infiltrate a demineralized nacre matrix with gelatin and to
form magnetite nanoparticles inside the gel matrix. Magnetization measurements Magnetic properties of the nanocomposite were measured by
using a superconducting quantum interference device (SQUID)
magnetometer. Figure 7 illustrates the magnetization loops
(magnetization M versus applied field H) of a representative
dried hybrid material with a mineral content of 65 wt % after
eight mineralization cycles at 293 K and 2 K. At T = 293 K the
hysteresis curve shows zero coercivity and zero remanence as it
is characteristic for superparamagnetic material [39] with a
particle size less than 20 nm. Due to magnetic anisotropy the
hysteresis curve at T = 2 K shows ferrimagnetic hysteresis. The
saturation magnetization for all analyzed samples is around
26 emu/g at 298 K and 36 emu/g at 2 K which are similar
values already reported before for the synthesis of gelatin-based
magnetic hydrogels [34]. Similar results can be obtained for the
analysis of magnetite nanoparticles prepared by a co-precipita-
tion method in water [40-42]. In order to determine the effect of
varying mineral content onto the magnetic properties, samples
with a particle load of 15 wt % to 65 wt % have been analyzed. For all analyzed samples similar results for the magnetic
hysteresis as well as for the saturation magnetization have
been obtained. Therefore, we conclude that the mineral
content as well as the transfer of the synthesis protocol to the Figure 6: SANS and VSANS scattering patterns of magnetite in
gelatin–chitin composite and of ferrogel in a mixed D2O/H2O solvent of
28 vol % D2O and 72 vol % H2O. The solid lines represent the fitting of
the Beaucage expression [30]. Figure 6: SANS and VSANS scattering patterns of magnetite in
gelatin–chitin composite and of ferrogel in a mixed D2O/H2O solvent of
28 vol % D2O and 72 vol % H2O. The solid lines represent the fitting of
the Beaucage expression [30]. Rg = 0.6 μm with Q−2 power law, which is characteristic for
chain-like clusters, are found at small Q. Individual magnetite
particles become visible at larger Q showing a slightly smaller
diameter of about D ≈ 18.5 nm (Rg = 7.2 nm). Thus, in the pres- Figure 7: Magnetic properties of the synthesized hybrid materials. a) Magnetization curves of a representative dried sample at 2 K and 293 K. Inset:
Enlargement of the low-field region showing the different coercive fields for the NPs at 2 and 293 K. SANS on magnetite formation in the
gelatin–chitin composite The magnetite–gelatin–chitin structure was characterized by
SANS contrast variation experiments, which is a beneficial
method to obtain information about the inorganic components
as well as the organic part. By using the matching point of
gelatin (28 vol % D2O) only the inorganic particles are visible
whereas the organic structure can be visualized working in pure
D2O. This technique is a standard tool in various fields
such as biomineralization [37,38]. Figure 6 demonstrates two
SANS–VSANS scattering profiles of magnetite in a 140 Beilstein J. Nanotechnol. 2015, 6, 134–148. ence of nacre organic matrix, the fiber-like chitin structure helps
with the formation of linearly aligned magnetite nanoparticles
(pearl-necklace-like, power law of Q−1), while in the gelatin gel
matrix without chitin, the nanoparticles exhibit a branch-like
arrangement (power law of Q−2). Figure 6: SANS and VSANS scattering patterns of magnetite in
gelatin–chitin composite and of ferrogel in a mixed D2O/H2O solvent of
28 vol % D2O and 72 vol % H2O. The solid lines represent the fitting of
the Beaucage expression [30]. Simulation studies Figure 8: Degree of sample swelling plotted as a function of the
swelling time at 23 °C for different samples with a gelatin concentra-
tion of 10 wt %. The equilibrium swelling degrees Sd (%) for the plotted
samples are 622.97 ± 88.31 (chitin–gelatin), 259.70 ± 38.46 (chitin
demineralized) and 121.94 ± 5.13 (chitin–gelatin–magnetite RC 6). Figure 8: Degree of sample swelling plotted as a function of the
swelling time at 23 °C for different samples with a gelatin concentra-
tion of 10 wt %. The equilibrium swelling degrees Sd (%) for the plotted
samples are 622.97 ± 88.31 (chitin–gelatin), 259.70 ± 38.46 (chitin
demineralized) and 121.94 ± 5.13 (chitin–gelatin–magnetite RC 6). In the case of nacre matrix infiltrated with gelatin a distinct
increase in swelling can be observed as compared to the insol-
uble matrix alone. This effect is not surprising as gelatin alone
shows a higher swelling capacity as the insoluble organic
matrix. The gravimetric water uptake of the gelatin–chitin
composite is similar to already reported swelling capacities of
gelatin. This observation is an additional proof for the
successful infiltration of gelatin inside the chitin layers. In the
case of the magnetic composite material, the swelling degree is
significantly decreased due to the presence of magnetite
nanoparticles, which act as additional crosslinkers in the gelatin
hydrogel. This effect was discovered before for the studies of
magnetic hydrogels [34] and shows similar values for the In the case of nacre matrix infiltrated with gelatin a distinct
increase in swelling can be observed as compared to the insol-
uble matrix alone. This effect is not surprising as gelatin alone
shows a higher swelling capacity as the insoluble organic
matrix. The gravimetric water uptake of the gelatin–chitin
composite is similar to already reported swelling capacities of
gelatin. This observation is an additional proof for the
successful infiltration of gelatin inside the chitin layers. In the
case of the magnetic composite material, the swelling degree is
significantly decreased due to the presence of magnetite
nanoparticles, which act as additional crosslinkers in the gelatin
hydrogel. This effect was discovered before for the studies of
magnetic hydrogels [34] and shows similar values for the Swelling studies In order to probe structural changes of the nanocomposite
during gelatin infiltration as well as during magnetite synthesis,
swelling studies were performed. The swelling capacity of the
insoluble nacre matrix, the gelatin infiltrated chitin matrix and
the magnetic nanocomposite are shown in Figure 8. The
swelling degree, Sd, is defined as: Magnetization measurements Attraction of modified nacre with b) no magnetic
field and c) external magnetic field (ca. 1 Tesla). Figure 7: Magnetic properties of the synthesized hybrid materials. a) Magnetization curves of a representative dried sample at 2 K and 293 K. Inset:
Enlargement of the low-field region showing the different coercive fields for the NPs at 2 and 293 K. Attraction of modified nacre with b) no magnetic
field and c) external magnetic field (ca. 1 Tesla). 141 Beilstein J. Nanotechnol. 2015, 6, 134–148. swelling degree. We can conclude that the gelatin hydrogel as
well as the magnetic hydrogel do not change their swelling
capacity inside the insoluble chitin matrix and therefore we
conclude that the structural changes are similar than the one for
already reported magnetic hydrogels. layered organic matrix does not affect the magnetic nature of
the material. Simulation studies To investigate the molecular scale interactions that account for
the formation of the magnetite–protein composite, we
performed molecular simulation studies of FeII(OH)2 and
FeIII(OH)3 motif association to two sets of biomolecular
matrices. To allow direct comparison to our previous study on
collagen-based composites [34,43], the association of an iron
hydroxide ion cluster to collagen (mimicked by a triple helix of
(Gly–Pro–Hyp)n peptides) is contrasted to ion association to
chitin. The latter model was chosen as three poly-(1,4)-D-
glucose chains of about 40 Å length (which corresponds to nine
monomers) stacked in three layers, which are connected by
hydrogen bonds. where Ws stands for the weight of the swollen sample after
swelling equilibrium was reached and Wd stands for the dry
weight before water uptake. Figure 8: Degree of sample swelling plotted as a function of the
swelling time at 23 °C for different samples with a gelatin concentra-
tion of 10 wt %. The equilibrium swelling degrees Sd (%) for the plotted
samples are 622.97 ± 88.31 (chitin–gelatin), 259.70 ± 38.46 (chitin
demineralized) and 121.94 ± 5.13 (chitin–gelatin–magnetite RC 6). As a starting point, the association of FeII(OH)2 and FeIII(OH)3
ion clusters was investigated in vacuum. From a series of
docking runs we found practically equivalent protein–ion
complexes for either collagen or chitin. However, the nature of
these complexes was found to differ significantly upon relax-
ation in aqueous solution. Figure 9 illustrates the association of
the two ion cluster types to collagen and typical configurations
as obtained from relaxation in aqueous solution based on 100 ps
molecular dynamics simulation runs. While the FeIII(OH)3 ion
clusters bind as stable moieties to the biomolecule, the associ-
ation of FeII(OH)2 to collagen was found to be less favored. Indeed, for 30% of the relaxation runs in aqueous solution the
latter cluster was observed to partially dissociate, which led to
(stable) FeII(OH)−–collagen complexes. In contrast to this, the
association of FeII(OH)2 and FeIII(OH)3 ion clusters to chitin
was found to be stable in both vacuum (Figure 10) and in
aqueous solution (Figure 11). As the FeII(OH)2 cluster reflects
an important motif of the magnetite structure we conclude that
our simulations show, at least from a qualitative point of view, a
slight preference of chitin over collagen as a nucleator for
magnetite [43]. Mechanical characterization To examine the mechanical properties of the composite ma-
terials we conducted some preliminary experiments. Force spec-
troscopy measurements with the colloidal probe technique
[46,47] were performed on bare and nanoparticle-loaded gelatin
as well as on bare and ferrogel loaded chitin scaffolds. From the
obtained force versus deformation curves we can already see
significant qualitative differences. Figure 12 shows a compari-
son of pure and nanoparticle-filled gelatin. With the addition of 142 Beilstein J. Nanotechnol. 2015, 6, 134–148. Figure 9: Representative structure of a triple helical (Gly–Hyp–Pro)n peptide [44] of 100 Å length with two associated iron clusters. a) The ferric ion
(light blue) is coordinated by seven oxygen atoms of which the three hydroxides show the strongest interaction and an Fe–O distance of 2.7 Å. The
Fe–O distances to the solvent and to carbonyl/hydroxy groups of collagen were found to be about 3 Å. b) The ferrous ion (green) is also coordinated
by seven oxygen atoms, but does not show a bipyramidal structure. More importantly, one of the hydroxide ions dissociated into the solvent. The
Fe–O distances for iron–collagen and iron–water contacts were found to be about 3 Å, whilst the remaining hydroxide ion exhibits an Fe–O distance
of 3.2 Å. Colors: Fe2+ (green), Fe3+ (light blue), O (red), H (white), N (dark blue), C (grey). Figure 10: Illustration of a β-chitin model [45] consisting of three poly-(1,4)-D-glucose chains of nine monomers stacked in three layers. the superparamagnetic particles the slope of the force curves
increases, i.e., the stiffness or mechanical resistance of the gels
is enhanced. This increase can be explained by the strength-
ening of the gelatin network by the rigid nanoparticles. These
have been shown to interact with the amide bonds along the
gelatin backbone [48] and might give rise to additional Figure 9: Representative structure of a triple helical (Gly–Hyp–Pro)n peptide [44] of 100 Å length with two associated iron clusters. a) The ferric ion
(light blue) is coordinated by seven oxygen atoms of which the three hydroxides show the strongest interaction and an Fe–O distance of 2.7 Å. The
Fe–O distances to the solvent and to carbonyl/hydroxy groups of collagen were found to be about 3 Å. b) The ferrous ion (green) is also coordinated
by seven oxygen atoms, but does not show a bipyramidal structure. More importantly, one of the hydroxide ions dissociated into the solvent. Mechanical characterization Figure 12: Force vs deformation characteristic of pure gelatin and
gelatin with ferromagnetic particles. Introduction of nanoparticles leads
to a significant increase of the stiffness of the material. Figure 13: Force vs deformation characteristics of the chitin scaffold
and the final composite. Introduction of ferrogel leads to a detectable
increase of the stiffness of the material. crosslinking. As a consequence, the flexibility of the gelatin
chains is reduced resulting in the observed stiffness increase
and the decreased swelling. Regarding the chitin scaffolds we
notice a stiffening effect as well (Figure 13). Introducing the
ferrogel reinforces the framework and gives the composite
superior mechanical performance. Nanoindentation testing with
AFM colloidal probe is a powerful technique as it combines
high lateral and force resolution with well-defined contact
geometry. It has successfully been applied to a range of systems
including capsules [49-52], full particles [53-55] and films [56-
59]. However, due to the morphological and structural inhomo-
geneity of our samples it is currently difficult to make a quanti-
tative evaluation of the data. Continuum mechanics models
typically require homogeneous and isotropic materials. For pure
gelatin we can successfully fit the obtained curves assuming the
Hertz model for a sphere in contact with a plane surface [60]
(see Supporting Information File 1). Thus, an elastic modulus of
2.6 ± 0.3 kPa is calculated which is in good agreement with data
from literature reporting modulus values in the low-kPa range
[58,61]. In contrast, the data from experiments on ferrogel or
composite show large scattering and the curves do not show a
shape that can be described by one of the established mechan-
ical theories. These deviations can be ascribed to the aforemen-
tioned non-ideal boundary conditions. It will be the aim of
future research to investigate the mechanical properties more
thoroughly. Figure 13: Force vs deformation characteristics of the chitin scaffold
and the final composite. Introduction of ferrogel leads to a detectable
increase of the stiffness of the material. mineral content of our hybrid material by repetition of reaction
cycles, the mineral content varies form 15 wt % (one reaction
cycle) to 65 wt % (eight reaction cycles). SQUID measure-
ments showed that our composite material shows superpara-
magnetic behavior, which is typical for magnetite nanoparticles
in this size range. Swelling studies indicate a structural change
of the gelatin inside the hybrid material upon introduction of the
magnetite nanoparticles. Mechanical characterization The
Fe–O distances for iron–collagen and iron–water contacts were found to be about 3 Å, whilst the remaining hydroxide ion exhibits an Fe–O distance
of 3.2 Å. Colors: Fe2+ (green), Fe3+ (light blue), O (red), H (white), N (dark blue), C (grey). Figure 9: Representative structure of a triple helical (Gly–Hyp–Pro)n peptide [44] of 100 Å length with two associated iron clusters. a) The ferric ion
(light blue) is coordinated by seven oxygen atoms of which the three hydroxides show the strongest interaction and an Fe–O distance of 2.7 Å. The
Fe–O distances to the solvent and to carbonyl/hydroxy groups of collagen were found to be about 3 Å. b) The ferrous ion (green) is also coordinated
by seven oxygen atoms, but does not show a bipyramidal structure. More importantly, one of the hydroxide ions dissociated into the solvent. The
Fe–O distances for iron–collagen and iron–water contacts were found to be about 3 Å, whilst the remaining hydroxide ion exhibits an Fe–O distance
of 3.2 Å. Colors: Fe2+ (green), Fe3+ (light blue), O (red), H (white), N (dark blue), C (grey). Figure 10: Illustration of a β-chitin model [45] consisting of three poly-(1,4)-D-glucose chains of nine monomers stacked in three layers. Figure 10: Illustration of a β-chitin model [45] consisting of three poly-(1,4)-D-glucose chains of nine monomers stacked in three layers. the superparamagnetic particles the slope of the force curves
increases, i.e., the stiffness or mechanical resistance of the gels
is enhanced. This increase can be explained by the strength- ening of the gelatin network by the rigid nanoparticles. These
have been shown to interact with the amide bonds along the
gelatin backbone [48] and might give rise to additional ening of the gelatin network by the rigid nanoparticles. These
have been shown to interact with the amide bonds along the
gelatin backbone [48] and might give rise to additional 143 Beilstein J. Nanotechnol. 2015, 6, 134–148. Figure 12: Force vs deformation characteristic of pure gelatin and
gelatin with ferromagnetic particles. Introduction of nanoparticles leads
to a significant increase of the stiffness of the material. Figure 12: Force vs deformation characteristic of pure gelatin and
gelatin with ferromagnetic particles. Introduction of nanoparticles leads
to a significant increase of the stiffness of the material. Figure 13: Force vs deformation characteristics of the chitin scaffold
and the final composite. Mechanical characterization By incorporation of more and more
inorganic material we can control the degree of swelling and
therefore the mechanical properties of the composite material. This result is supported by preliminary AFM colloidal probe
measurements. Simulation studies show the binding of iron and
hydroxide ions to both collagen and β-chitin. Direct compari-
son, however, indicates that chitin should be the more favored
nucleator macromolecule species for magnetite thus boosting
composite growth along the chitin fibers. Mechanical characterization Introduction of ferrogel leads to a detectable
increase of the stiffness of the material. Figure 11: a) Representative structure for the coordination of
FeIII(OH)3 by chitin. The ferric ion (light blue) is coordinated by four
different types of oxygen atoms (red) forming seven coordinative inter-
actions. b) Coordination of FeII(OH)2 by chitin exhibiting a stable coor-
dination by both hydroxide ions of the ion cluster. In summary, seven
oxygen atoms coordinate the ferrous ion (green) building a pentagonal
bipyramid, with the cluster hydroxide oxygens building the tops with a
distance of 2.86 Å. The pentagonal plane consists of two oxygen
atoms from solvent molecules forming weaker bonds of 3.1 Å and
three protein contacts, whereby one carbonyl oxygen atom binds over
2.9 Å and two hydroxy oxygens over 3.1 Å. Figure 11: a) Representative structure for the coordination of
FeIII(OH)3 by chitin. The ferric ion (light blue) is coordinated by four
different types of oxygen atoms (red) forming seven coordinative inter-
actions. b) Coordination of FeII(OH)2 by chitin exhibiting a stable coor-
dination by both hydroxide ions of the ion cluster. In summary, seven
oxygen atoms coordinate the ferrous ion (green) building a pentagonal
bipyramid, with the cluster hydroxide oxygens building the tops with a
distance of 2.86 Å. The pentagonal plane consists of two oxygen
atoms from solvent molecules forming weaker bonds of 3.1 Å and
three protein contacts, whereby one carbonyl oxygen atom binds over
2.9 Å and two hydroxy oxygens over 3.1 Å. Figure 11: a) Representative structure for the coordination of
FeIII(OH)3 by chitin. The ferric ion (light blue) is coordinated by four
different types of oxygen atoms (red) forming seven coordinative inter-
actions. b) Coordination of FeII(OH)2 by chitin exhibiting a stable coor-
dination by both hydroxide ions of the ion cluster. In summary, seven
oxygen atoms coordinate the ferrous ion (green) building a pentagonal
bipyramid, with the cluster hydroxide oxygens building the tops with a
distance of 2.86 Å. The pentagonal plane consists of two oxygen
atoms from solvent molecules forming weaker bonds of 3.1 Å and
three protein contacts, whereby one carbonyl oxygen atom binds over
2.9 Å and two hydroxy oxygens over 3.1 Å. Figure 12: Force vs deformation characteristic of pure gelatin and
gelatin with ferromagnetic particles. Introduction of nanoparticles leads
to a significant increase of the stiffness of the material. Sample characterizations Samples of Coomassie-stained thin cuts were observed under
bright field transmission mode by using a Zeiss optical micro-
scope equipped with a video camera (AxioCam MRc5). Fluo-
rescent labeled samples were analyzed with a confocal fluores-
cence laser scanning microscope (Zeiss LSM 510 Meta) at an
excitation wavelength of 543 nm. Chemicals Microtome cuts of embedded samples were incubated with
0.2 wt % Coomassie blue G-250 (Sigma-Aldrich) at room
temperature for 2 h. After washing with acetic acid the cuts
were carefully washed three times with destaining solution
(30% ethanol/60% water/10% acetic acid). The following commercially available chemicals were
purchased and applied in the syntheses without further purifica-
tion: FeCl2·4H2O (Sigma-Aldrich), FeCl3·6H2O (Sigma-
Aldich), 0.1 M NaOH solution (Merck), gelatin type B (~225
Bloom, Sigma-Aldrich), 4-chloro-m-cresol (Fluka), methanol
(VWR). For the preparation of the reactant solutions double-
distilled and deionized (Milli-Q) water was used. All solutions
were degassed with argon before usage. Gelatin preparation The gelatin hydrogels were prepared as described elsewhere
[34]. Here briefly, different amounts of gelatin powder were
mixed with water and the gelatin granules were allowed to swell
for 24 h at 6 °C. In order to obtain a homogeneous gel, the
swollen mixture is heated for at least 2 h at 50 °C. 20 mL of the
gelatin sol are filled into crystallization dishes and left at room
temperature for gelation. In order to avoid bacterial growth, a
5 wt % solution of 4-chloro-m-cresol in methanol was added
(0.15 mL per 1 g of gelatin granules). In situ synthesis of magnetite nanoparticles In situ mineralization of magnetite nanoparticles inside the
gelatin hydrogel chitin composite material was carried out
through co-precipitation of FeCl2 and FeCl3 after an already
established synthesis protocol [34]. Briefly, the gelatin chitin
composite sample was introduced into a solution, containing
FeCl2 (0.1 M) and FeCl3 (0.2 M), where it was left for 96 h at
6 °C. The iron(II)- and iron(III)-loaded matrix was washed with
water and placed in 0.1 M NaOH solution for 150 min. Preparation of insoluble organic nacre matrix Shells of Haleotis laevigata were sand-blasted to remove the
calcite layer. After thorough washing with deionized water, the
shells were dried overnight at room temperature and cut into
pieces with an area of around 1 cm × 1 cm. The nacre pieces
were demineralized with 10 vol % acetic acid and solvent
exchange every day for at least 5 d. The remaining organic
matrix was washed with Milli-Q water until neutral pH was
reached. Rhodamine B ITC staining Microtome cuts of embedded demineralized nacre matrix
were incubated at 60 °C with 0.1 wt % rhodamine B ITC
(Sigma-Aldrich) in water for 3 h. After washing with water the
cuts were accurately washed with acidified ethanol for three
times. Conclusion In summary, we have developed a synthetic method to fabri-
cate a multifunctional hybrid material. We can successfully
infiltrate gelatin into the insoluble nacre matrix and synthesize
magnetite nanoparticles inside our template. We can control the In summary, we have managed to synthesize a bio-inspired
organic–inorganic hybrid material, which combines the struc- 144 Beilstein J. Nanotechnol. 2015, 6, 134–148. pump. Vacuum was then applied until bubbling of the solution
was observed. The vacuum was then removed to force the
liquid gelatin to be drawn into the tissue. The whole process
was repeated for three times. After gelatin infiltration the nacre
matrix pieces were left inside the gelatin-filled crystallization
dishes and allowed to stand for gelation first 5 h at room
temperature and finally kept at 6 °C for 24 h before further
usage. For further processing the gelatin-filled insoluble
organic nacre parts were cut out of the gelatin hydrogel with a
scalpel. tural features of nacre and chiton teeth. Swelling studies and
preliminary mechanical measurements indicate a change in the
mechanical properties as compared to pure gelatin. This is
controllable through the adjustable mineral content. In combina-
tion with the superparamagnetic behavior, we have therefore
generated a material with improved mechanical performance
coupled with magnetic properties. More quantitative future
mechanical measurements will show in how far the fracture
resistance of nacre could be combined with the wear resistance
of chiton teeth. Supporting Information Supporting Information File 1
Additional experimental data. [http://www.beilstein-journals.org/bjnano/content/
supplementary/2190-4286-6-13-S1.pdf] Supporting Information File 1
Additional experimental data. [http://www.beilstein-journals.org/bjnano/content/
supplementary/2190-4286-6-13-S1.pdf] Infiltration of gelatin inside the insoluble
nacre matrix For TEM examination the formed composite material was dehy-
drated with a graded ethanol series and embedded in LR White
Resin (Medium Grade). The sample was cut perpendicular with
a diamond knife in a Leica ultracut UCT and transferred onto a
Formvar-coated copper grid. TEM and electron diffraction were
performed on a Zeiss Libra 120 operating at 120 kV. For SEM
measurements the samples were air-dried at room temperature
and cut perpendicular to the chitin layers with a scalpel. The The cut demineralized insoluble organic nacre pieces are put
into crystallization dishes filled with 20 mL liquid gelatin at
55 °C. To maintain uniform contact of the matrix pieces with
the hot gelatin solution a filter paper covered the liquid surface
to prevent floating. The complete set-up was then placed into a
vacuum desiccator and the desiccator was attached to a vacuum 145 Beilstein J. Nanotechnol. 2015, 6, 134–148. series of 200 independent docking runs were performed for
each ionic species. sample was placed on a sticky carbon tape and coated with a
thin layer of gold in order to avoid charging effects. The SEM
measurements were performed on Zeiss Neon 40 EsB oper-
ating in high vacuum. An InLens and SE detector was used for
signal collection and an acceleration voltage of 5 kV was
chosen for recording the images. Simulation studies Molecular Simulation: as described in [34] a series of
FeIII(OH)x(OH2)4−x and FeII(OH)y(OH2)6−y clusters were pre-
modeled from ab-initio calculations in vacuum. For all clusters
high-spin constellation was identified as preferred by several
electron volts. Imposing overall charge neutrality (i.e.,
x + y = 3 + 2) we found the neutral FeIII(OH)3·(H2O) and the
FeII(OH)2·4(H2O) as energetically preferred. Docking to
collagen and chitin was modeled in aqueous solution by using
empirical force fields [44,45,63,64]. Investigation of bio-
logically designed metal-specific chelators for potential metal
recovery and waste remediation applications [65], and the
Kawska–Zahn docking procedure were described previously
[43]. Acknowledgements Many thanks go to A. Laptev (University of Konstanz) for
performing the SQUID measurements, Dr. Joachim Hentschel
and Lauretta Nejedli for microtomy services (Electron
Microscopy Center at the University of Konstanz). Fluores-
cence microscopy images were acquired at the Bioimaging
Center at the University of Konstanz (BIC). This work was
supported by the Deutsche Forschungsgemeinschaft within the
priority program SPP 1569 "Generation of Multifunctional Inor-
ganic Materials by Molecular Bionics". Mechanical characterization Force spectroscopy experiments were conducted at the atomic
force microscope (AFM) Nanowizard® I (JPK Instruments,
Berlin, Germany) in a custom-built liquid cell (diameter 2 cm,
height 0.5 cm). Thin slices (1–2 mm) of swollen hydrogels were
cut from the bulky samples with a scalpel and immobilized at
the bottom of the cell by using two component epoxy glue
(UHU Endfest 300, UHU GmbH & Co. KG, Bühl, Germany). All measurements were performed in Milli-Q-water at room
temperature. As a probe a tipless silicon nitride cantilever (NSC
12, no Al coating, MikroMasch, Tallinn, Estonia) was used with
a glass sphere (35 µm in diameter, Polysciences Europe GmbH,
Eppelheim, Germany) attached to its front (colloidal probe). Before the actual measurements, the cantilevers were calibrated
against the non-deformable glass substrate to determine their
optical lever sensitivity resulting as the slope of the recorded
force–displacement curve. The deformation of the sample was
obtained by subtraction of the bending of the cantilever from
the raw displacement data. The spring constant of the cantilever
(0.56 N/m) was deduced from its thermal noise spectrum prior
to the attachment of the colloidal probe [66]. Small-angle neutron scattering (SANS and VSANS): SANS and
VSANS experiments were carried out at the KWS1 and KWS 3
diffractometers operated by Jülich Center for Neutron Research
(JCNS) at the Forschungs-Neutronenquelle Heinz Maier-Leib-
nitz (FRM II) in Garching, Germany [62]. Some of the SANS
data at large Q range is based on experiments performed at the
SANS II, Swiss spallation neutron source SINQ, Paul Scherrer
Institute, Villigen, Switzerland. The mineral content of the multifunctional hybrid material was
determined by means of TGA (Netzsch, Selb, Germany). Measurements were carried out at a heating rate of 5 K/min
under a constant oxygen flow. Samples were scanned from
293 K to 1273 K. Magnetization measurements were carried out by using a
quantum design superconducting quantum interference device
(SQUID) 5 T magnetic properties measurement system
(MPMS). For measurements, dried samples were introduced
into gelatin capsules and magnetization loop measurements at
2 K and 293 K were performed. Supporting Information
Supporting Information File 1
Additional experimental data. [http://www.beilstein-journals.org/bjnano/content/
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English
| null |
Supplemental Table 4 from Race-associated Molecular Changes in Gynecologic Malignancies
| null | 2,023
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cc-by
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Supplementary Table 6 Supplementary Table 6 chr
pos
strand
Name
UCSC RefGene
Name
adj.P.Val
∆Beta
Tumor
chrX
15130
6718
+
cg04950711
MAGEA10;MA
GEA10
0.02
-0.29
OV
chr7
10054
4704
-
cg00644033
0.01
-0.28
OV
chrX
52840
736
-
cg25993152
XAGE5
0.02
-0.27
OV
chr1
0
44881
551
+
cg00499822
CXCL12;CXCL
12;CXCL12
0.00
-0.21
OV
chr1
9
29011
47
-
cg08634464
ZNF57
0.01
-0.20
OV
chr6
41130
718
+
cg20095587
TREM2
0.05
-0.19
OV
chr9
11790
5075
+
cg04761824
1-Dec
0.01
-0.18
OV
chr1
9
42260
485
-
cg26813458
CEACAM6
0.02
-0.17
OV
chr7
99573
278
+
cg19465374
AZGP1
0.03
-0.17
OV
chrX
15130
7053
-
cg19964192
MAGEA10;MA
GEA10
0.00
-0.16
OV
chr1
4
96152
109
+
cg27504299
TCL1B;TCL1B
0.02
-0.16
OV
chr2
0
57427
830
+
cg17414107
GNAS;GNAS;G
NAS
0.02
-0.16
OV
chr1
15925
8877
-
cg14696870
FCER1A
0.04
-0.16
OV
chr1
9
15123
14
-
cg04601137
ADAMTSL5
0.02
-0.15
OV
chr5
54281
336
-
cg20451680
ESM1;ESM1
0.03
-0.15
OV
chr4
69111
580
+
cg19510180
TMPRSS11B
0.02
-0.15
OV
chr6
32731
327
-
cg04345908
HLA-DQB2
0.03
-0.15
OV
chr2
95537
475
-
cg05723825
TEKT4
0.00
-0.15
OV
chrX
11837
0356
-
cg07876586
PGRMC1
0.00
-0.15
OV
chr2
0
57427
738
+
cg27661264
GNAS;GNAS;G
NAS
0.03
-0.14
OV
chr3
66550
735
+
cg26131019
LRIG1
0.00
-0.14
OV
chr6
11001
1156
-
cg01500097
AKD1;FIG4;AK
D1
0.05
-0.13
OV
chr1
7
39197
721
-
cg02022375
KRTAP1-1
0.01
-0.13
OV
chr1
1
55703
443
-
cg25890048
OR5I1
0.01
-0.12
OV
chr1
9
10736
038
-
cg17826679
SLC44A2;SLC4
4A2
0.03
-0.12
OV chr6
10976
0831
+
cg20811607
PPIL6;PPIL6;S
MPD2
0.01
-0.11
OV
chr1
20305
9178
-
cg13626881
0.04
-0.11
OV
chr2
1
45705
686
-
cg17356252
AIRE
0.02
-0.11
OV
chrX
10074
0937
+
cg14457691
ARMCX4
0.05
-0.10
OV
chr1
35220
814
-
cg01333788
GJB5
0.02
-0.10
OV
chr2
23900
9036
-
cg09039163
ESPNL;ESPNL
0.02
-0.10
OV
chrX
69487
197
-
cg11494656
ARR3
0.04
-0.10
OV
chrX
41783
155
-
cg16269097
CASK;CASK;C
ASK
0.03
-0.10
OV
chrX
15208
6723
-
cg16022279
ZNF185
0.01
-0.10
OV
chrX
15344
8957
+
cg06325687
OPN1MW2;OP
N1MW
0.03
-0.10
OV
chr2
1
36421
472
+
cg04915566
RUNX1;RUNX1
0.01
0.11
OV
chr1
2
15373
987
-
cg19205533
RERG
0.03
0.14
OV
chr1
1
85780
971
+
cg01120308
PICALM;PICAL
M
0.03
0.14
OV
chr1
6
56642
024
+
cg17886959
MT2A
0.01
0.14
OV
chr3
11185
2156
+
cg25462303
GCET2;GCET2
0.02
0.14
OV
chr1
16303
9012
-
cg22605643
RGS4;RGS4;R
GS4;RGS4
0.04
0.17
OV
chr1
2
15114
703
-
cg10925082
ARHGDIB
0.03
0.20
OV
chr1
4
39735
211
+
cg13277939
CTAGE5;CTAG
E5;CTAGE5;CT
AGE5
0.04
0.23
OV
chr1
9
49249
932
-
cg10234985
IZUMO1;IZUM
O1
0.05
0.26
OV
chr1
9
29011
47
-
cg08634464
ZNF57
5.30E-20
-0.34
BRCA
chr1
1
65405
362
-
cg25361844
SIPA1
6.79E-05
-0.29
BRCA
chr3
13925
8939
+
cg23363832
RBP1;RBP1;RB
P1
1.22E-04
-0.25
BRCA
chr8
23083
353
-
cg26530341
TNFRSF10A
5.20E-10
-0.24
BRCA
chr5
11869
1033
-
cg07086380
TNFAIP8;TNFA
IP8
1.06E-08
-0.23
BRCA
chr3
66550
735
+
Cg
26131019
LRIG1
4.62E-88
-0.22
BRCA
chr1
5
48936
953
-
cg18671950
FBN1
2.53E-02
-0.22
BRCA chr9
90113
813
-
cg15746719
DAPK1
9.82E-08
-0.21
BRCA
chr2
20209
8951
-
cg26799474
CASP8;CASP8;
CASP8;CASP8
5.35E-04
-0.19
BRCA
chr8
10979
9756
+
cg15042080
TMEM74;TME
M74
5.17E-03
-0.19
BRCA
chr3
14283
8847
-
cg00995327
CHST2;CHST2
2.95E-02
-0.17
BRCA
chr1
1
44330
903
+
cg26365854
ALX4
3.26E-03
-0.16
BRCA
chr1
7
33701
321
-
cg18108623
SLFN11;SLFN1
1;SLFN11;SLF
N11;SLFN11
3.45E-02
-0.16
BRCA
chr1
0
64575
798
-
cg19355190
EGR2;EGR2;E
GR2;EGR2;EG
R2
1.25E-02
-0.16
BRCA
chr1
6
14302
16
-
cg11911951
UNKL
4.38E-04
-0.16
BRCA
chr1
7
25798
973
-
cg05246522
KSR1
2.42E-02
-0.15
BRCA
chr3
49378
032
+
cg18886444
USP4;USP4
1.29E-05
-0.15
BRCA
chr1
1
44327
399
-
cg04970352
ALX4
1.18E-02
-0.14
BRCA
chr1
0
12969
1429
+
cg23817637
CLRN3
4.08E-17
-0.14
BRCA
chr1
4
85996
495
+
cg17410236
FLRT2;FLRT2
2.44E-02
-0.14
BRCA
chr1
5
10141
9074
-
cg23191950
ALDH1A3
5.06E-06
-0.14
BRCA
chr1
1
65405
903
-
cg26668713
SIPA1
6.72E-06
-0.14
BRCA
chr4
48485
233
-
cg19283196
SLC10A4
3.37E-02
-0.14
BRCA
chr8
13377
2889
+
cg20955688
TMEM71;TME
M71;TMEM71;T
MEM71
3.05E-04
-0.13
BRCA
chr2
0
56286
697
+
cg00138126
PMEPA1;PMEP
A1;PMEPA1
2.68E-04
-0.13
BRCA
chr1
9
33863
876
-
cg15046693
CEBPG
1.00E-05
-0.13
BRCA
chr1
0
82049
429
+
cg19423196
MAT1A;MAT1A
4.97E-03
-0.13
BRCA
chr3
12045
459
-
cg15873301
SYN2;SYN2
8.05E-05
-0.13
BRCA
chr5
76114
373
-
cg24573501
F2RL1
2.52E-03
-0.13
BRCA
chr2
23900
9036
-
cg09039163
ESPNL;ESPNL
5.30E-20
-0.13
BRCA
chr1
7
80186
273
-
cg14417329
SLC16A3;SLC1
6A3
3.20E-12
-0.13
BRCA
chr7
49813
763
-
cg00333226
VWC2
6.08E-03
-0.13
BRCA
chr1
9
67391
92
-
cg02085507
TRIP10
1.85E-02
-0.13
BRCA chr8
82192
606
+
cg19904463
FABP5
9.78E-06
-0.13
BRCA
chr1
25255
838
+
cg00117172
RUNX3;RUNX3
2.27E-03
-0.13
BRCA
chr8
12598
4927
+
cg23968383
ZNF572
3.71E-08
-0.13
BRCA
chr9
36037
454
+
cg12717594
RECK
3.69E-03
-0.12
BRCA
chr1
7
46800
639
-
cg01543654
C17orf93;PRAC
2.07E-02
-0.12
BRCA
chr2
46523
461
-
cg17518825
EPAS1
2.60E-14
-0.12
BRCA
chr2
43019
614
-
cg20289949
HAAO
1.52E-02
-0.12
BRCA
chr1
9
35981
677
-
cg10305797
KRTDAP
1.77E-05
-0.12
BRCA
chr1
3
78492
916
-
cg23316360
EDNRB;EDNR
B;EDNRB;EDN
RB;EDNRB
9.00E-03
-0.12
BRCA
chr1
4
76045
348
+
cg04001333
FLVCR2
1.32E-08
-0.12
BRCA
chr1
8
28473
09
+
cg09009111
EMILIN2
1.73E-02
-0.12
BRCA
chr1
7
27369
780
+
cg06144905
PIPOX
6.44E-03
-0.12
BRCA
chr6
11079
7497
+
cg22334665
SLC22A16
4.34E-03
-0.12
BRCA
chr9
34263
021
+
cg19947621
KIF24
1.77E-04
-0.12
BRCA
chr1
76540
465
-
cg12601757
ST6GALNAC3;
ST6GALNAC3;
ST6GALNAC3;
ST6GALNAC3
3.40E-02
-0.12
BRCA
chr9
90114
156
-
cg24754277
DAPK1
6.56E-04
-0.12
BRCA
chr8
27850
178
+
cg07634191
SCARA5;SCAR
A5
1.78E-05
-0.12
BRCA
chr7
23510
648
-
cg02860543
IGF2BP3
3.39E-02
-0.11
BRCA
chr3
13925
8822
+
cg06543018
RBP1;RBP1;RB
P1
9.24E-08
-0.11
BRCA
chr6
15177
2946
-
cg00510787
C6orf211;RMN
D1
2.60E-14
-0.11
BRCA
chr2
10830
764
-
cg02196655
NOL10
1.42E-04
-0.11
BRCA
chr1
7
39197
721
-
cg02022375
KRTAP1-1
5.68E-08
-0.11
BRCA
chr3
71834
640
+
cg08555612
PROK2;PROK2
6.97E-03
-0.11
BRCA
chr1
15879
9935
-
cg25119415
MNDA
6.20E-03
-0.11
BRCA
chr1
7
27944
585
-
cg06038133
CORO6
1.79E-03
-0.11
BRCA
chr1
2
11245
2424
-
cg26789453
TMEM116;ERP
29;ERP29
3.34E-03
-0.11
BRCA chr1
5
10141
9017
-
cg19224278
ALDH1A3
4.36E-04
-0.11
BRCA
chr2
0
53092
781
+
cg11822659
DOK5
4.40E-06
-0.11
BRCA
chr8
13377
2742
+
cg27159719
TMEM71;TME
M71;TMEM71;T
MEM71
2.10E-03
-0.11
BRCA
chr1
9
39687
963
+
cg19917856
NCCRP1
1.03E-02
-0.11
BRCA
chr1
23404
0833
-
cg25437385
SLC35F3
4.24E-02
-0.11
BRCA
chr3
14893
9523
-
cg17439694
CP
2.96E-11
-0.11
BRCA
chr1
20315
6246
-
cg07423149
CHI3L1
1.22E-02
-0.11
BRCA
chr4
11155
7894
-
cg05522383
PITX2;PITX2
9.50E-03
-0.11
BRCA
chr1
2
75728
469
-
cg21087137
GLIPR1L1;GLIP
R1L1
6.41E-03
-0.10
BRCA
chr1
7
10102
558
-
cg13991233
GAS7
2.30E-03
-0.10
BRCA
chr8
41165
699
-
cg02388150
SFRP1
6.32E-04
-0.10
BRCA
chr3
44902
933
-
cg16615211
MIR564;TMEM
42
1.90E-02
-0.10
BRCA
chr1
7
66166
53
-
cg16652063
SLC13A5;SLC1
3A5;SLC13A5;
SLC13A5
2.89E-02
-0.10
BRCA
chr1
20581
9179
+
cg14159672
PM20D1
8.63E-11
-0.10
BRCA
chr1
17181
0972
-
cg23391785
DNM3;DNM3
6.27E-04
-0.10
BRCA
chr1
8
56530
302
+
cg12406559
ZNF532;ZNF53
2
3.21E-02
-0.10
BRCA
chr1
3
47469
654
+
cg00308665
HTR2A;HTR2A
1.49E-03
0.10
BRCA
chr1
9
66397
3
+
cg03605761
RNF126
1.39E-06
0.10
BRCA
chr8
25281
609
+
cg22502171
GNRH1;GNRH
1;GNRH1
1.98E-06
0.10
BRCA
chr1
6
30406
122
+
cg16014085
ZNF48
2.45E-03
0.10
BRCA
chr1
9
14920
43
-
cg02674804
REEP6
5.63E-05
0.10
BRCA
chr3
12233
4227
-
cg22468055
PARP15;PARP
15
1.65E-05
0.10
BRCA
chr6
29455
532
+
cg01078434
MAS1L
3.47E-02
0.10
BRCA
chr1
6
15527
940
-
cg07977490
C16orf45
3.87E-09
0.10
BRCA
chr4
26493
496
-
cg04353483
CCKAR
1.77E-02
0.10
BRCA
chr1
8
48189
538
-
cg26946769
MAPK4
5.74E-03
0.10
BRCA chr1
8
63417
392
+
cg04564030
CDH7;CDH7
2.87E-02
0.11
BRCA
chr2
2
37916
157
+
cg22506059
CARD10
3.19E-08
0.11
BRCA
chr1
7
46622
491
-
cg09313705
HOXB2
2.21E-04
0.11
BRCA
chr1
9
58262
242
-
cg02682905
ZNF776
6.63E-04
0.11
BRCA
chr2
0
43935
361
+
cg21835643
MATN4;MATN4
;RBPJL;MATN4
3.59E-02
0.11
BRCA
chr1
47155
20
-
cg17525406
AJAP1;AJAP1
3.61E-02
0.11
BRCA
chr2
0
44657
948
+
cg22752533
SLC12A5;SLC1
2A5;SLC12A5
1.29E-03
0.11
BRCA
chr8
80523
461
-
cg23326689
STMN2
4.31E-06
0.11
BRCA
chr7
64126
140
+
cg12856392
ZNF107;ZNF10
7
5.26E-05
0.11
BRCA
chr1
20332
0223
-
cg03764585
FMOD;FMOD
3.40E-06
0.11
BRCA
chr4
58950
98
+
cg22680204
CRMP1
1.44E-02
0.11
BRCA
chr1
7
66950
545
-
cg21660392
ABCA8
2.92E-03
0.11
BRCA
chr3
11236
0952
+
cg21307628
CCDC80;CCDC
80
2.72E-04
0.11
BRCA
chr7
89840
731
+
cg27626102
STEAP2;STEA
P2
4.71E-05
0.11
BRCA
chr5
24645
487
-
cg01058368
CDH10
1.24E-06
0.11
BRCA
chr3
18738
8225
+
cg13206017
SST
1.48E-03
0.11
BRCA
chr5
36301
436
-
cg26511075
RANBP3L;RAN
BP3L
2.37E-04
0.11
BRCA
chr6
31088
188
+
cg08424423
CDSN;PSORS1
C1
7.08E-06
0.11
BRCA
chr2
18254
4413
-
cg02836529
NEUROD1
5.08E-04
0.11
BRCA
chr2
0
15386
71
-
cg25737664
SIRPD
1.89E-02
0.11
BRCA
chr1
6
60688
35
-
cg19378133
A2BP1;A2BP1
2.18E-03
0.11
BRCA
chr1
15100
9588
-
cg11584936
BNIPL;BNIPL
5.26E-03
0.11
BRCA
chr1
15328
3994
-
cg06275635
PGLYRP3
1.73E-02
0.11
BRCA
chr1
6
31214
417
-
cg12100791
PYCARD;PYCA
RD
1.45E-03
0.11
BRCA
chr1
0
17171
695
-
cg10707565
CUBN
2.27E-03
0.11
BRCA
chr4
16900
199
-
cg08899626
LDB2;LDB2;LD
B2;LDB2
4.64E-03
0.11
BRCA
chr4
68324
0
+
cg17952262
MFSD7
9.54E-05
0.12
BRCA chr3
18656
0617
-
cg03573747
ADIPOQ
4.38E-05
0.12
BRCA
chr1
33160
791
-
cg05342835
SYNC;SYNC
6.32E-11
0.12
BRCA
chr1
10416
0257
+
cg22215192
AMY2A
4.85E-06
0.12
BRCA
chr3
86937
06
+
cg22959932
C3orf32;C3orf3
2
2.16E-05
0.12
BRCA
chr3
58523
313
+
cg13705284
ACOX2
1.80E-08
0.12
BRCA
chr6
16727
5395
-
cg13217373
RPS6KA2
7.81E-07
0.12
BRCA
chr8
64205
06
-
cg24670715
ANGPT2;ANGP
T2;ANGPT2;AN
GPT2;ANGPT2;
ANGPT2;MCPH
1
5.15E-05
0.12
BRCA
chr2
0
39928
631
-
cg22294908
ZHX3;ZHX3
1.16E-02
0.12
BRCA
chr1
20631
7403
+
cg21478437
CTSE;CTSE
1.23E-04
0.12
BRCA
chr5
14058
8299
+
cg07899016
PCDHB12;PCD
HB12
7.69E-04
0.12
BRCA
chr1
11867
308
-
cg14472778
CLCN6;CLCN6;
CLCN6;CLCN6;
MTHFR
1.48E-05
0.12
BRCA
chr9
10424
8926
+
cg06912252
C9orf125
5.46E-03
0.12
BRCA
chr1
7
36004
61
+
cg13246592
P2RX5;P2RX5;
P2RX5
3.48E-04
0.12
BRCA
chr1
1
57148
215
-
cg24459209
PRG3
1.20E-05
0.12
BRCA
chr1
0
80946
79
+
cg04765277
FLJ45983;FLJ4
5983
3.33E-03
0.12
BRCA
chr1
2
49449
136
+
cg13007988
MLL2
1.71E-03
0.12
BRCA
chr1
2
11463
844
+
cg22692158
PRB4
4.19E-06
0.12
BRCA
chr7
11405
5419
+
cg05232889
FOXP2;FOXP2;
FOXP2
4.55E-04
0.12
BRCA
chr6
30659
643
+
cg16979445
NRM
1.62E-03
0.12
BRCA
chr1
1
64624
52
+
cg11547724
HPX
8.66E-07
0.12
BRCA
chr6
16672
2773
+
cg05089968
PRR18
2.93E-05
0.12
BRCA
chr5
95467
55
-
cg03702236
SEMA5A
7.33E-06
0.12
BRCA
chr1
2
21525
662
-
cg15583072
SLCO1A2;IAPP
9.54E-05
0.12
BRCA
chr1
18362
2007
-
cg20579480
APOBEC4;RGL
1
1.46E-05
0.12
BRCA
chr1
4
23653
098
+
cg27035169
SLC7A8
1.30E-11
0.12
BRCA chr1
1
63272
554
+
cg11484576
LGALS12;LGAL
S12;LGALS12
1.93E-06
0.13
BRCA
chr7
87855
714
+
cg02399455
SRI
1.45E-03
0.13
BRCA
chr2
0
58507
171
+
cg07347645
SYCP2;SYCP2
2.87E-02
0.13
BRCA
chr1
29852
18
+
cg08528984
FLJ42875;PRD
M16;PRDM16;F
LJ42875
8.57E-03
0.13
BRCA
chr1
7
48546
118
-
cg06958829
ACSF2;CHAD
2.60E-08
0.13
BRCA
chr8
14412
0106
-
cg07770222
C8orf31
1.52E-03
0.13
BRCA
chr1
2
53208
762
+
cg12610744
KRT4
5.06E-06
0.13
BRCA
chr2
16734
3774
+
cg05331214
SCN7A
3.49E-03
0.13
BRCA
chr1
15516
1784
-
cg24512973
MUC1;MUC1;M
UC1;MUC1;MU
C1;MUC1;MUC
1
5.45E-09
0.13
BRCA
chr2
12122
3909
-
cg17204557
LOC84931
1.06E-07
0.13
BRCA
chr8
13205
4555
-
cg13912117
8.58E-03
0.13
BRCA
chr1
19723
8398
+
cg00321478
CRB1
1.55E-04
0.13
BRCA
chr2
2
42896
688
+
cg12078929
SERHL
4.76E-07
0.13
BRCA
chr1
7
79424
06
-
cg15799267
ALOX15B;ALO
X15B;ALOX15B
;ALOX15B;ALO
X15B;ALOX15B
7.56E-04
0.13
BRCA
chr2
0
58533
443
+
cg20895028
CDH26
1.88E-04
0.13
BRCA
chr2
22028
3175
+
cg18182399
DES;DES
7.15E-05
0.13
BRCA
chr3
11395
7903
-
cg03109316
ZNF80
1.83E-02
0.13
BRCA
chr2
11969
9682
-
cg11009736
MARCO
1.67E-05
0.14
BRCA
chr1
20703
9302
-
cg23282674
IL20
2.02E-04
0.14
BRCA
chr5
95769
008
+
cg23187653
PCSK1
8.52E-04
0.14
BRCA
chr1
0
10892
3781
-
cg16415058
SORCS1;SOR
CS1
8.75E-08
0.14
BRCA
chr2
18373
1407
+
cg20308679
FRZB;FRZB
8.79E-06
0.14
BRCA
chr1
20161
7042
+
cg25167447
NAV1
9.82E-08
0.14
BRCA
chr1
7
48637
104
-
cg05942574
CACNA1G;CAC
NA1G;CACNA1
G;CACNA1G;C
ACNA1G;CACN
8.28E-05
0.14
BRCA A1G;CACNA1G
;CACNA1G;CA
CNA1G;CACNA
1G;CACNA1G;
CACNA1G;CAC
NA1G;CACNA1
G;CACNA1G
chr1
22601
2913
-
cg24928687
EPHX1;EPHX1
7.86E-04
0.14
BRCA
chr2
0
58533
495
-
cg24607535
CDH26;CDH26
1.57E-03
0.14
BRCA
chr1
9
15052
824
-
cg03544379
OR7C2
3.09E-03
0.14
BRCA
chr1
16330
784
-
cg24525573
C1orf64;C1orf6
4
9.69E-05
0.14
BRCA
chr1
4
99177
777
-
cg06906435
C14orf177
6.04E-03
0.15
BRCA
chr1
0
80946
41
-
cg00779924
FLJ45983;FLJ4
5983
1.96E-03
0.15
BRCA
chr1
7
33795
67
+
cg02228185
ASPA;ASPA
9.39E-07
0.15
BRCA
chr1
15301
3830
-
cg14826683
SPRR2D
1.13E-03
0.15
BRCA
chr1
1
91124
71
-
cg01081263
SCUBE2;SCUB
E2
4.06E-09
0.15
BRCA
chr1
0
26223
310
-
cg23771603
MYO3A
6.06E-03
0.15
BRCA
chr1
9
52391
250
+
cg16731240
ZNF577;ZNF57
7;ZNF577
1.87E-06
0.15
BRCA
chr3
19531
0883
-
cg05624196
APOD;APOD
2.39E-05
0.16
BRCA
chr5
13707
1523
+
cg13847070
KLHL3;KLHL3
3.06E-05
0.16
BRCA
chr1
18494
4288
-
cg25182523
FAM129A
1.94E-07
0.16
BRCA
chr1
1
26743
425
+
cg05953243
SLC5A12;SLC5
A12
4.59E-04
0.16
BRCA
chr7
86973
677
-
cg15350036
CROT;CROT;C
ROT;TP53TG1
5.74E-06
0.16
BRCA
chr6
56112
291
-
cg13830624
COL21A1;COL
21A1
2.35E-02
0.16
BRCA
chr1
20305
8954
-
cg11719784
2.99E-04
0.17
BRCA
chr1
8
55400
167
-
cg18085435
ATP8B1
4.21E-05
0.17
BRCA
chr1
2
38615
67
+
cg17904739
EFCAB4B;EFC
AB4B;EFCAB4
B
1.03E-09
0.17
BRCA
chr4
76555
547
-
cg14988503
CDKL2;CDKL2
3.41E-02
0.17
BRCA
chr3
18409
5505
+
cg11819637
THPO
6.77E-07
0.17
BRCA chr1
5
45803
341
+
cg08057475
SLC30A4;C15o
rf21
5.84E-04
0.17
BRCA
chr2
15728
0411
-
cg24579667
4.93E-05
0.18
BRCA
chr1
17301
9720
-
cg19589427
TNFSF18
3.09E-06
0.18
BRCA
chr1
7
48546
620
-
cg06818777
CHAD;ACSF2
5.86E-07
0.18
BRCA
chr1
1
26744
467
-
cg20092728
SLC5A12
5.91E-03
0.18
BRCA
chr1
8
70535
925
+
cg21922574
NETO1
2.66E-06
0.18
BRCA
chr6
50786
670
+
cg25593948
TFAP2B
1.99E-04
0.18
BRCA
chr5
13944
491
-
cg03503295
DNAH5
8.39E-06
0.18
BRCA
chr1
6
56672
415
-
cg09137696
MT1A
1.20E-03
0.19
BRCA
chr4
14117
8469
+
cg04457051
SCOC;SCOC
3.04E-10
0.19
BRCA
chr1
2
11062
035
-
cg08658594
TAS2R13;TAS2
R13;PRR4;PRH
1
7.08E-08
0.19
BRCA
chr6
13736
6322
+
cg22487322
IL20RA
4.97E-16
0.20
BRCA
chr1
6
28674
34
-
cg22730830
PRSS21;PRSS
21;PRSS21
2.26E-19
0.21
BRCA
chr1
20332
0386
-
cg26987645
FMOD
8.72E-14
0.22
BRCA
chr1
2
64837
39
-
cg18738906
SCNN1A;SCNN
1A;SCNN1A
9.84E-06
0.23
BRCA
chr1
7
49295
615
-
cg02880679
MBTD1
3.04E-06
0.23
BRCA
chr1
24802
0812
-
cg07533148
TRIM58
1.24E-04
0.27
BRCA
chr1
16825
0628
+
cg17095936
TBX19
9.72E-08
0.35
BRCA
chr1
0
13504
4114
+
cg09053680
UTF1
0.03
-0.48
UCEC
chr6
73332
073
-
cg24687051
KCNQ5;KCNQ5
;KCNQ5;KCNQ
5;KCNQ5
0.00
-0.48
UCEC
chr2
68546
507
-
cg07080358
CNRIP1;CNRIP
1
0.01
-0.45
UCEC
chr1
1
20690
957
+
cg17371081
NELL1;NELL1
0.01
-0.43
UCEC
chr1
8
55438
01
+
cg00027083
EPB41L3
0.03
-0.39
UCEC
chr1
8
49866
548
+
cg21669679
DCC;DCC
0.01
-0.36
UCEC
chr4
16587
8091
-
cg00393585
C4orf39;TRIM6
1
0.01
-0.35
UCEC
chr1
2
85306
916
+
cg03064067
SLC6A15;SLC6
A15;SLC6A15
0.00
-0.34
UCEC chr1
4
38677
218
+
cg27590397
SSTR1;SSTR1
0.01
-0.33
UCEC
chr1
8
74962
369
+
cg04534765
GALR1;GALR1
0.02
-0.32
UCEC
chr1
8
74961
787
+
cg10486998
GALR1
0.03
-0.32
UCEC
chr1
4
27066
634
-
cg18488855
NOVA1;NOVA1
;NOVA1
0.02
-0.31
UCEC
chr5
16097
5024
+
cg01939681
GABRB2;GABR
B2
0.01
-0.31
UCEC
chr1
9
29011
47
-
cg08634464
ZNF57
0.00
-0.30
UCEC
chr1
0
17796
67
+
cg05307923
ADARB2;ADAR
B2
0.01
-0.30
UCEC
chr1
6
85932
853
-
cg24826867
IRF8
0.02
-0.30
UCEC
chr1
8
74961
785
+
cg15343119
GALR1
0.03
-0.30
UCEC
chr2
21022
565
-
cg09109450
C2orf43
0.00
-0.28
UCEC
chr1
8
63417
392
+
cg04564030
CDH7;CDH7
0.04
-0.28
UCEC
chr4
66535
145
+
cg18420965
EPHA5;EPHA5
0.01
-0.26
UCEC
chr1
9
46477
338
+
cg21461100
NOVA2
0.01
-0.26
UCEC
chr1
8
74961
727
+
cg26721264
GALR1
0.03
-0.26
UCEC
chr2
1
31312
091
+
cg09542111
GRIK1;GRIK1;
GRIK1;GRIK1
0.02
-0.26
UCEC
chr2
0
53092
781
+
cg11822659
DOK5
0.01
-0.25
UCEC
chr2
45169
447
+
cg13163729
SIX3
0.01
-0.25
UCEC
chr1
1
13178
0332
-
cg20881910
NTM;NTM;NTM
;NTM
0.02
-0.25
UCEC
chr1
2
85306
474
-
cg14449051
SLC6A15;SLC6
A15;SLC6A15;
SLC6A15;SLC6
A15;SLC6A15
0.02
-0.25
UCEC
chr1
0
12807
6936
+
cg11668923
ADAM12;ADAM
12;ADAM12;AD
AM12
0.05
-0.25
UCEC
chr3
12045
459
-
cg15873301
SYN2;SYN2
0.01
-0.24
UCEC
chr6
73331
652
+
cg15717808
KCNQ5;KCNQ5
;KCNQ5;KCNQ
5;KCNQ5;KCN
Q5;KCNQ5;KC
NQ5;KCNQ5;K
CNQ5
0.01
-0.24
UCEC
chr1
53528
612
-
cg23092823
PODN
0.05
-0.23
UCEC
chr8
24815
006
+
cg13266631
NEFL
0.02
-0.23
UCEC chr5
45695
922
-
cg06498267
HCN1
0.01
-0.22
UCEC
chr4
42285
71
+
cg14882700
OTOP1
0.02
-0.22
UCEC
chr1
15890
1938
+
cg19884600
PYHIN1;PYHIN
1;PYHIN1;PYHI
N1
0.03
-0.22
UCEC
chr7
75245
6
+
cg13577076
PRKAR1B;PRK
AR1B;PRKAR1
B;PRKAR1B;P
RKAR1B;PRKA
R1B
0.01
-0.22
UCEC
chr1
8
31020
511
-
cg05245861
C18orf34;C18or
f34
0.02
-0.21
UCEC
chr2
1
31312
328
-
cg21816539
GRIK1;GRIK1
0.01
-0.21
UCEC
chr8
73450
181
+
cg20890210
KCNB2
0.04
-0.21
UCEC
chr1
0
12807
7323
+
cg13488201
ADAM12;ADAM
12
0.04
-0.21
UCEC
chr1
8
74961
424
+
cg12699371
GALR1
0.03
-0.21
UCEC
chr1
6
10277
017
+
cg25047001
GRIN2A;GRIN2
A;GRIN2A
0.05
-0.20
UCEC
chr6
62996
697
+
cg02217159
KHDRBS2
0.01
-0.19
UCEC
chr1
1
13178
0946
-
cg11965370
NTM;NTM;NTM
;NTM;NTM;NT
M;NTM
0.01
-0.19
UCEC
chr3
21792
434
+
cg18267381
ZNF385D;ZNF3
85D
0.05
-0.19
UCEC
chr1
38230
769
+
cg12128017
EPHA10;EPHA
10;EPHA10;EP
HA10
0.04
-0.19
UCEC
chr1
3
27334
259
+
cg22179082
GPR12
0.04
-0.19
UCEC
chr1
0
10360
3722
+
cg25123470
KCNIP2;KCNIP
2;KCNIP2;KCNI
P2;KCNIP2;KC
NIP2
0.05
-0.19
UCEC
chrX
91034
003
-
cg07474356
PCDH11X
0.02
-0.19
UCEC
chr1
0
10108
8930
-
cg01968793
CNNM1;CNNM
1
0.01
-0.19
UCEC
chr1
8
74963
364
-
cg00662556
GALR1
0.01
-0.19
UCEC
chr3
19190
202
-
cg07657236
KCNH8;KCNH8
0.05
-0.18
UCEC
chr2
19305
9405
+
cg06856528
TMEFF2;TMEF
F2
0.01
-0.18
UCEC
chr1
8
49868
101
+
cg25602457
DCC
0.04
-0.18
UCEC
chr2
0
59826
207
-
cg15534366
CDH4
0.00
-0.18
UCEC
chr1
24802
+
cg20855565
TRIM58
0.02
-0.18
UCEC 0350
chr5
15178
4441
+
cg03914397
NMUR2
0.04
-0.18
UCEC
chr1
9
44905
343
-
cg09030119
ZNF285A
0.02
-0.18
UCEC
chr1
9
50831
901
-
cg06572160
KCNC3
0.04
-0.18
UCEC
chr2
11960
6055
-
cg03266453
EN1
0.05
-0.18
UCEC
chr8
13205
4555
-
cg13912117
0.02
-0.17
UCEC
chr3
16491
4437
-
cg13619915
SLITRK3;SLITR
K3
0.01
-0.17
UCEC
chr8
13916
4635
+
cg06276653
FAM135B
0.01
-0.17
UCEC
chr7
86273
429
-
cg14724613
GRM3;GRM3
0.04
-0.17
UCEC
chr1
5
35046
760
+
cg21053529
GJD2
0.02
-0.17
UCEC
chr1
7
80186
273
-
cg14417329
SLC16A3;SLC1
6A3
0.00
-0.17
UCEC
chrX
55478
180
+
cg18869368
MAGEH1
0.03
-0.17
UCEC
chr1
1
13340
2230
+
cg03923934
OPCML;OPCM
L
0.03
-0.17
UCEC
chr3
66550
735
+
cg26131019
LRIG1
0.00
-0.17
UCEC
chr1
3
84456
127
-
cg07104706
SLITRK1;SLITR
K1
0.01
-0.17
UCEC
chr1
8
74964
030
-
cg05896682
GALR1
0.02
-0.16
UCEC
chr3
62359
420
+
cg06423920
FEZF2
0.01
-0.16
UCEC
chr9
10450
0729
-
cg08997253
GRIN3A;GRIN3
A
0.00
-0.16
UCEC
chr1
35672
14
-
cg19135761
WDR8
0.05
-0.16
UCEC
chr7
76038
785
-
cg13594711
SRCRB4D;SRC
RB4D;ZP3
0.02
-0.16
UCEC
chr1
9
44905
882
-
cg26918645
ZNF285A
0.05
-0.16
UCEC
chr2
22904
6325
-
cg04072323
SPHKAP;SPHK
AP;SPHKAP;S
PHKAP
0.01
-0.16
UCEC
chr1
1
59855
616
-
cg22197708
MS4A2;MS4A2
0.01
-0.16
UCEC
chr3
16491
4471
-
cg14717170
SLITRK3
0.02
-0.15
UCEC
chr1
1
21615
86
-
cg25163476
INS-
IGF2;IGF2;IGF2
AS;IGF2;IGF2A
S;IGF2
0.05
-0.15
UCEC
chr1
23720
5098
+
cg19764418
RYR2
0.01
-0.15
UCEC chr1
20622
3241
+
cg18992688
AVPR1B
0.00
-0.15
UCEC
chr1
9
56510
892
-
cg05023116
NLRP5
0.03
-0.15
UCEC
chr1
1
10548
1509
+
cg19343464
GRIA4;GRIA4;
GRIA4;GRIA4;
GRIA4
0.01
-0.15
UCEC
chr4
90757
533
+
cg26578617
SNCA;SNCA;S
NCA;SNCA
0.05
-0.15
UCEC
chrX
14099
1592
+
cg25651984
MAGEC1
0.01
-0.15
UCEC
chr4
15612
9725
-
cg27504805
NPY2R
0.04
-0.15
UCEC
chr2
11037
3002
+
cg03552103
SEPT10;SEPT1
0;ANKRD57
0.01
-0.15
UCEC
chr9
10450
1030
+
cg18794577
GRIN3A
0.01
-0.14
UCEC
chr1
23685
0232
-
cg21376883
ACTN2
0.04
-0.14
UCEC
chr1
2
51532
88
+
cg20792062
KCNA5;KCNA5
0.01
-0.14
UCEC
chr1
0
26223
100
+
cg08441170
MYO3A;MYO3
A
0.00
-0.14
UCEC
chr9
12213
2261
+
cg25935911
DBC1
0.01
-0.14
UCEC
chr1
0
50817
306
+
cg18592174
SLC18A3;CHA
T
0.02
-0.14
UCEC
chr1
3
70682
324
-
cg20523861
KLHL1;KLHL1;
ATXN8OS
0.03
-0.14
UCEC
chr1
35220
814
-
cg01333788
GJB5
0.00
-0.14
UCEC
chr1
8
44336
399
-
cg19751300
ST8SIA5
0.04
-0.13
UCEC
chr1
9
41195
910
+
cg08450982
NUMBL
0.02
-0.13
UCEC
chr1
9
31770
476
-
cg08458170
TSHZ3
0.01
-0.13
UCEC
chr1
0
12969
1429
+
cg23817637
CLRN3
0.02
-0.13
UCEC
chr1
7
79060
98
-
cg25465406
GUCY2D
0.05
-0.13
UCEC
chrX
85303
309
-
cg25488547
CHM;CHM
0.02
-0.13
UCEC
chr1
1
11334
6327
+
cg12758687
DRD2;DRD2
0.05
-0.13
UCEC
chr6
78173
250
-
cg08614481
HTR1B
0.02
-0.13
UCEC
chr7
30833
38
+
cg26937500
CARD11;CARD
11
0.01
-0.12
UCEC
chr1
66258
441
-
cg26832142
PDE4B;PDE4B
0.00
-0.12
UCEC
chr4
14755
9579
-
cg13262687
POU4F2
0.03
-0.12
UCEC
chr7
13013
1258
+
cg13917504
MEST;MEST;M
EST;MESTIT1;
0.03
-0.12
UCEC MEST
chr3
45092
68
-
cg18847227
SUMF1;SUMF1
;SUMF1
0.02
-0.12
UCEC
chr1
37499
309
+
cg06722633
GRIK3
0.04
-0.12
UCEC
chr1
8
56887
785
-
cg23357981
GRP;GRP;GRP
0.04
-0.12
UCEC
chrX
14489
9412
-
cg27584469
SLITRK2;SLITR
K2;SLITRK2;SL
ITRK2;SLITRK2
;SLITRK2;SLIT
RK2;SLITRK2
0.02
-0.12
UCEC
chr1
9
57349
815
+
cg19771589
PEG3;PEG3;ZI
M2;PEG3;PEG
3;ZIM2;ZIM2
0.02
-0.12
UCEC
chr2
0
30225
517
-
cg27413508
COX4I2
0.02
-0.12
UCEC
chr6
13356
1932
-
cg20330472
EYA4;EYA4;EY
A4
0.00
-0.12
UCEC
chr8
32405
427
-
cg19162158
NRG1;NRG1;N
RG1;NRG1;NR
G1;NRG1;NRG
1;NRG1;NRG1;
NRG1;NRG1;N
RG1
0.02
-0.11
UCEC
chr2
46523
461
-
cg17518825
EPAS1
0.00
-0.11
UCEC
chr1
2
45542
95
-
cg01731341
FGF6
0.03
-0.11
UCEC
chr5
14448
78
+
cg26205131
SLC6A3
0.01
-0.11
UCEC
chr7
49813
763
-
cg00333226
VWC2
0.05
-0.11
UCEC
chr1
0
26223
310
-
cg23771603
MYO3A
0.01
-0.11
UCEC
chr5
44389
282
+
cg20387341
FGF10
0.04
-0.11
UCEC
chrX
75647
859
-
cg04544498
MAGEE1
0.02
-0.11
UCEC
chr1
7
39867
328
+
cg12229387
GAST
0.00
-0.11
UCEC
chr2
73520
043
-
cg13481359
EGR4
0.03
-0.11
UCEC
chr2
23900
9036
-
cg09039163
ESPNL;ESPNL
0.00
-0.10
UCEC
chr1
4
24804
022
+
cg12265829
ADCY4
0.02
-0.10
UCEC
chr6
11130
2729
-
cg05213296
RPF2
0.01
-0.10
UCEC
chr1
9
42800
54
-
cg18581445
SHD;SHD
0.01
-0.10
UCEC
chr1
6
71264
290
-
cg20977864
HYDIN;HYDIN
0.05
-0.10
UCEC
chr9
13070
0866
-
cg23006204
DPM2
0.03
-0.10
UCEC chr1
1
20173
62
+
cg22172494
H19
0.01
0.10
UCEC
chr1
7
67137
953
-
cg22081096
ABCA6;ABCA6
0.02
0.10
UCEC
chr9
13096
8072
+
cg05826823
CIZ1;DNM1;DN
M1;CIZ1
0.02
0.11
UCEC
chr5
85110
1
+
cg18429742
ZDHHC11
0.01
0.11
UCEC
chr1
1
20187
24
+
cg07342901
H19;MIR675
0.02
0.11
UCEC
chr4
26493
496
-
cg04353483
CCKAR
0.04
0.11
UCEC
chr9
13993
9792
+
cg26581729
NPDC1
0.05
0.11
UCEC
chr6
15295
8899
-
cg27316956
SYNE1;SYNE1
0.00
0.11
UCEC
chr1
7
42092
431
+
cg12259256
TMEM101
0.02
0.11
UCEC
chr2
2
37216
067
+
cg02978737
PVALB
0.01
0.12
UCEC
chr1
1
20176
64
-
cg25852472
H19
0.02
0.12
UCEC
chr2
2
38201
690
+
cg07141002
H1F0
0.05
0.12
UCEC
chr2
22028
3175
+
cg18182399
DES;DES
0.01
0.13
UCEC
chr1
7
34345
136
+
cg14916288
CCL23;CCL23
0.02
0.13
UCEC
chr1
1
80600
60
-
cg15480475
TUB
0.01
0.13
UCEC
chr1
2
38615
67
+
cg17904739
EFCAB4B;EFC
AB4B;EFCAB4
B
0.01
0.13
UCEC
chr8
11814
6418
-
cg23338195
SLC30A8
0.03
0.16
UCEC
chr2
1
33957
290
+
cg20857253
TCP10L
0.03
0.17
UCEC
chr1
1
20174
83
-
cg26808784
H19
0.00
0.19
UCEC
chrX
28821
83
+
cg22376897
ARSE
0.00
0.24
UCEC
chrX
28823
33
+
cg11964613
ARSE;ARSE
0.00
0.25
UCEC
chr3
12316
7276
-
cg13878010
ADCY5
0.01
0.29
UCEC
chr3
66550
735
+
cg26131019
LRIG1
5.74E-14
-0.23
CESC
chr2
46523
461
-
cg17518825
EPAS1
0.005
-0.15
CESC
Chr: chromosome of probe-associated gene
Pos: Genomic base position UCSC RefGene Name
adj.P.Val: P value adjusted for multiple hypothesis testing with BH method
delta Beta: Difference in mean beta methylation level (EA – AA)
Tumor: CESC = cervix, UCEC = uterine endometrial carcinoma, OV = ovary, BRCA =
breast UCSC RefGene Name
adj.P.Val: P value adjusted for multiple hypothesis testing with BH method
delta Beta: Difference in mean beta methylation level (EA – AA)
Tumor: CESC = cervix, UCEC = uterine endometrial carcinoma, OV = ovary, BRCA =
breast UCSC RefGene Name
adj.P.Val: P value adjusted for multiple hypothesis testing with BH method
delta Beta: Difference in mean beta methylation level (EA – AA)
Tumor: CESC = cervix, UCEC = uterine endometrial carcinoma, OV = ovary, BRCA =
breast
|
https://openalex.org/W2031389288
|
https://www.epj-conferences.org/articles/epjconf/pdf/2012/19/epjconf_meso2012_09031.pdf
|
English
| null |
Study of heavy hyperons production in DIS at COMPASS
|
EPJ web of conferences
| 2,012
|
cc-by
| 2,166
|
1 Introduction It is important to measure the yields of heavy hyperons and antihyperons in deep inelastic scattering
(DIS) for for understanding the quark hadronization models. The decays of the heavy hyperons are
also important for Λ and ¯Λ production and polarization. It is known [1] that a significant part of
the Λ hyperons in DIS is produced indirectly, via decays of heavy hyperons such as Σ0, Σ(1385), Ξ
etc. The polarization of these indirect Λ hyperons may influence on the Λ polarization. On the other
hand, the role of the indirect ¯Λ hyperons, forming in the decays of heavy antihyperons, is not known. No measurements of the heavy antihyperon yields in the DIS exist before. Our measurements are
also demonstrated similar hyperon and antihyperon yields in DIS of muons. The yields of the heavy
hyperons in the neutrino DIS were measured by the NOMAD collaboration [2]. They have found
strong deviation of the measured yields from the Monte Carlo predictions. The experimental yields
were used to tune the parameters of the LEPTO generator. Study of heavy hyperons production in DIS at COMPASS N. Rossiyskaya on behalf of the COMPASS Collaborationa Joint Institute for Nuclear Research, Dubna, Russia Abstract. The yields of heavy hyperons and antihyperons have been studied in deep
inelastic scattering at the COMPASS experiment at CERN. The data were collected in
2003-2004 using a 160 GeV polarised muon beam scattered offa large polarised 6LiD
target. Signals from Σ+(1385), Σ−(1385), Ξ−(1321) and their antiparticles were recon-
structed. The ratios of Σ±/Λ, ¯Σ±/ ¯Λ, Ξ−/Λ and ¯Ξ+/ ¯Λ were determined. These ratios were
used to tune parameters of the LEPTO generator. Comparison with the yields of the heavy
hyperons at low Q2 - region was done. a e-mail: Natalia.Rossiyskaya@cern.ch This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which permit
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20123709031 2 The experimental set-up COMPASS (COmmon Muon and Proton Apparatus for Structure and Spectroscopy) is the fixed target
facility at CERN. The COMPASS spectrometer is designed to reconstruct the scattered muons and
the produced hadrons in wide momentum and angular ranges. It is divided in two stages with two
dipole magnets, SM1 and SM2. The first magnet, SM1, accepts charged particles of momenta larger
than 0.4 GeV/c, and the second one, SM2, those larger than 4 GeV/c. The angular acceptance of the
spectrometer is limited by the aperture of the polarised target magnet. For the upstream end of the
target it is ±70 mrad. To match the expected particle flux at various locations in the spectrometer,
COMPASS uses various tracking detectors. The identification of charged particles is possible with a
RICH detector, although in this paper we have not utilised the information from the RICH. The data
recording system is activated by various triggers indicating the presence of a scattered muon and an
energy deposited by hadrons in the calorimeters. More information on the COMPASS spectrometer
can be found in [3]. Study of heavy hyperons production in DIS at COMPASS
N. Rossiyskaya on behalf of the COMPASS Collaborationa
Joint Institute for Nuclear Research, Dubna, Russia
EPJ Web of Conferences
DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 2012
,
epjconf 20123
/
09031 (2012)
37
709031 DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 2012
epjconf 20123
/
709031 EPJ Web of Conferences
DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 2012
,
epjconf 20123
/
09031 (2012)
37
709031 EPJ Web of Conferences
DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 2012
,
epjconf 20123
/
09031 (2012)
37
709031 EPJ Web of Conferences
DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 2012
,
epjconf 20123
/
09031 (2012)
37
709031 3 The data analysis The data used in the present analysis were collected in 2003-2004 using a 160 GeV polarised muon
beam scattered offa large polarised 6LiD target. The beam intensity is 2×108 muons per spill. The
event selection requires a reconstructed interaction vertex defined by the incoming and the scat-
tered muon located inside the target. DIS events are selected by cuts on the photon virtuality (Q2 >
1 (GeV/c)2) and on the fractional energy of the virtual photon (0.2 < y < 0.9). The data sample
consists of 8.67 · 107 DIS events from the 2003 run and 22.5 · 107 DIS events from the 2004 run. We studied following decays of heavy hyperons and antihyperons: µ+ + d →µ+ + Σ±(1385) + X (Σ± →Λ + π±) (1) and ( ¯Σ± →¯Λ + π±) (2)
µ+ + d →µ+ + Ξ−(1321) + X (Ξ−→Λ + π−) (3) and ( ¯Ξ+ →¯Λ + π+) (4) Main selection criteria for reactions (1) and (2) require that the outcoming pion track is from the
primary vertex and the vector of the Λ( ¯Λ) momentum is pointing to the primary vertex. However, in the
weak hyperon (antihyperon) decays, reactions (3)-(4), a heavy hyperon decays in a secondary vertex. For these weak decays neither the pion track comes from the primary vertex nor the Λ( ¯Λ) momentum
points to the primary vertex. Main selection criteria for reactions (1) and (2) require that the outcoming pion track is from the
primary vertex and the vector of the Λ( ¯Λ) momentum is pointing to the primary vertex. However, in the
weak hyperon (antihyperon) decays, reactions (3)-(4), a heavy hyperon decays in a secondary vertex. For these weak decays neither the pion track comes from the primary vertex nor the Λ( ¯Λ) momentum
points to the primary vertex. The analysis is started from the reconstruction of the vector of the momentum of the Λ( ¯Λ) hyperon. A detailed description of the selection cuts is given [4]. The total number of events after all selection
cuts are N(Λ) = 99667 ± 385 and N( ¯Λ) = 60056 ± 322. This is much more then in all previous
experiments on Λ and ¯Λ production in DIS [1,2,5]. The Σ±(1385) and ¯Σ±(1385) resonances decay into the Λπ± and ¯Λπ± in the primary vertex. The
invariant mass distributions of Λπ± and ¯Λπ± from the experimental data are shown in Fig. 1, 2. Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20123709031 EPJ Web of Conferences MESON2012 - 12th International Workshop on Meson Production, Properties and Interaction These ratios are corrected on the experimental
acceptance evaluated using the MC data. The experimental relative yields (5)-(10) were used to tune the parameters of the LEPTO generator. The COMPASS Monte Carlo code is based on the LEPTO 6.5.1 generator [6] providing DIS events
which are passed through a GEANT-based apparatus simulation programme and the same chain of
reconstruction procedures as the experimental events. It turns out that the experimental yields of Λ
and ¯Λ hyperons differ from the MC ones by a factor of about 2. The yields of heavy hyperons and
antihyperons are also different for the data and the MC. That was the reason to tune the LEPTO
parameters to describe the present data. The parameters tuning are described in detail in [7,8]. p
p
p
g
It is important to know how the yields of hyperons change if one release the DIS cuts (Q2 >
1 (GeV/c)2 and 0.2 < y < 0.9). After removal of these cuts the data sample increased by a factor of 10. The total number of N was found to be N(Λ) = 1208413±1312 and N(Λ) = 654387±1067. However,
no changes in the ratios Σ/Λ, ¯Σ/ ¯Λ, Ξ/Λ and ¯Ξ/ ¯Λ was found within the statistical errors. Summarizing, the yields of the Σ(1385), ¯Σ(1385), Ξ(1321) and ¯Ξ(1321) hyperons are measured in
DIS induced by the muon beam. The percentage of the indirect Λ from the decays of Σ(1385), Ξ(1321)
in the total Λ sample is similar to the percentage of indirect ¯Λ from decays of ¯Σ(1385), ¯Ξ(1321). The
measured hyperon yields were used to tune the LEPTO generator parameters. After the removal of the
DIS cuts the ratios of the Σ/Λ, ¯Σ/ ¯Λ, Ξ/Λ, and ¯Ξ/ ¯Λ yields stays unchanged within the statistical errors. 1. HERMES Collaboration, A. Airapetyan et al.,, Phys. Rev. D74 (2006) 072004.
2. NOMAD Collaboration, P. Astier et al., Nucl.Phys. B621 (2002) 3.
3. COMPASS Collaboration, P.Abbon et al., Nucl. Instrum. Meth. A577 (2007) 455.
4. COMPASS Collaboration, M.Alekseev et al., Eur. Phys. J. C64 (2009) 171.
5. E665 Collaboration, M.R.Adams et al., Eur. Phys. J. C17 (2000) 263.
6. A.E.G.Ingelman, and J.Rathsman, Comp. Phys. Commun. 101, 10.
7. T. Sj¨ostrand, Comp. Phys. Commun. 82 (1994) 74.
8. COMPASS Collaboration, N.Rossiyskaya et al., Nucl.Phys. B (Proc.Suppl.), (2011) 39-42. MESON2012 - 12th International Workshop on Meson Production, Properties and Interaction MESON2012 - 12th International Workshop on Meson Production, Properties and Interaction 012 - 12th International Workshop on Meson Production, Properties and Interaction To select the weak hyperon decay reactions (3-4) the collinear cut for Λ( ¯Λ) hyperons is no longer
used. For this reason the collinearity cut θcol < 0.01 rad was omitted. It was substituted by the two
dimensional Closest Distance of Approach (CDA) procedure. The Ξ−(1321) and ¯Ξ+(1321) resonances
decay into the Λπ−and ¯Λπ+ in the secondary vertex. The invariant mass distributions of Λπ−and ¯Λπ+
from experimental data are shown in Fig. 3. 2
), GeV/c
−
π
+
Λ
M(
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
1.65
1.7
Events
0
100
200
300
400
500
600
deuteron 2003−2004
2
>1 (GeV/c)
2
Q
52
±
(1321)=1634
−
Ξ
N of
deuteron 2003−2004
2
>1 (GeV/c)
2
Q
52
±
(1321)=1634
−
Ξ
N of
2
), GeV/c
+
π
+
Λ
M(
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
1.65
1.7
Events
0
50
100
150
200
250
300
350
400
deuteron 2003−2004
2
>1 (GeV/c)
2
Q
44
±
(1321)=1054
+
Ξ
N of
deuteron 2003−2004
2
>1 (GeV/c)
2
Q
44
±
(1321)=1054
+
Ξ
N of
Fig. 3. Invariant mass distributions of Λπ−(left) and ¯Λπ+ (right) in the secondary vertex. Fig. 3. Invariant mass distributions of Λπ−(left) and ¯Λπ+ (right) in the secondary vertex. The numbers of events after the fit were found to be N(Ξ−) = 1634 ± 52 and N( ¯Ξ+) = 1054 ± 44. The ratios for the yields of the heavy (anti)hyperons production to Λ( ¯Λ) yields are the following: The numbers of events after the fit were found to be N(Ξ−) = 1634 ± 52 and N( ¯Ξ+) = 1054 ± 44. The ratios for the yields of the heavy (anti)hyperons production to Λ( ¯Λ) yields are the following: Σ+(1385)/Λ = 0.055 ± 0.005 ± 0.005 (5)
¯Σ−(1385)/ ¯Λ = 0.047 ± 0.006 ± 0.005 (6)
Σ−(1385)/Λ = 0.056 ± 0.009 ± 0.007 (7)
¯Σ+(1385)/ ¯Λ = 0.039 ± 0.006 ± 0.006 (8)
Ξ−(1321)/Λ = 0.037 ± 0.003 ± 0.002 (9)
¯Ξ+(1321)/ ¯Λ = 0.046 ± 0.004 ± 0.002 (10) Where the first error is statistic and second is systematic. These ratios are corrected on the experimental
acceptance evaluated using the MC data. Where the first error is statistic and second is systematic. 3 The data analysis In
Fig. 2 the broad peak on the right corresponds to the Σ−(1385) and ¯Σ+(1385) hyperons. The narrow
peak on the left is a part of the Ξ−(1321) and ¯Ξ+(1321) weak decays (3-4) passed through the selection
criteria. 2
), GeV/c
+
π
+
Λ
M(
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
Events
0
500
1000
1500
2000
2500
deuteron 2003−2004
2
>1 (GeV/c)
2
Q
333
±
(1385) = 3631
+
Σ
N of
2
), GeV/c
−
π
+
Λ
M(
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
Events
0
200
400
600
800
1000
1200
1400
1600
1800
deuteron 2003−2004
2
>1 (GeV/c)
2
Q
222
±
(1385) = 2173
−
Σ
N of
Fig. 1. Invariant mass distributions of Λπ+(left) and ¯Λπ−(right) in the primary vertex. Fig. 1. Invariant mass distributions of Λπ+(left) and ¯Λπ−(right) in the primary vertex. 2
), GeV/c
−
π
+
Λ
M(
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
Events
0
500
1000
1500
2000
2500
deuteron 2003−2004
2
>1 (GeV/c)
2
Q
490
±
(1385) = 2970
−
Σ
N of
2
), GeV/c
+
π
+
Λ
M(
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
Events
0
200
400
600
800
1000
1200
1400
1600
1800
2000
deuteron 2003−2004
2
>1 (GeV/c)
2
Q
265
±
(1385) = 1889
+
Σ
N of
Fig. 2. Invariant mass distributions of Λπ−(left) and ¯Λπ+ (right) in the primary vertex. Fig. 2. Invariant mass distributions of Λπ−(left) and ¯Λπ+ (right) in the primary vertex. The numbers of events after the fit were found to be N(Σ+) = 3631 ± 311, N(Σ−) = 2970 ± 490,
N( ¯Σ−) = 2173 ± 222 and N( ¯Σ+) = 1889 ± 265. 09031-p.2 09031-p.2 References 09031-p.3 09031-p.3
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https://zenodo.org/record/8363593/files/CAD-CAM%20DESIGN%20AND%20THE%20GENETIC%20OPTIMIZATION%20OF%20FEEDERS%20FOR%20SAND%20CASTING%20PROCESS.pdf
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CAD/CAM DESIGN AND GENETIC OPTIMIZATION OF FEEDERS FOR SAND CASTING PROCESS
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Facta Universitatis. Series: Mechanical Engineering
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cc-by
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FACTA UNIVERSITATIS
Series: Mechanical Engineering Vol. 14, No 2, 2016, pp. 147 - 158
Original scientific paper
CAD/CAM DESIGN AND THE GENETIC OPTIMIZATION
OF FEEDERS FOR SAND CASTING PROCESS
UDC 621.7
Nedeljko Dučić1, Žarko Ćojbašić2, Radomir Radiša3,
Radomir Slavković1, Ivan Milićević1
1
Faculty of Technical Sciences, Ĉaĉak, University of Kragujevac
2
Faculty of Mechanical Engineering, University of Niš
3
Research and Development Institute Lola L.T.D.
Abstract. The paper proposes methodology of feeder design and optimization for sand
casting process. Casting part is a part of excavator buckets, i.e. holder of the cutting
tooth. Process of design and optimization is based on the application of the rules, which
are the result of many years of work researchers in the field of metal casting. Computer
Aided Design (CAD) is used as a methodology in the design of feeders. Genetic Algorithm
(GA) as an artificial intelligence technique is used in the optimization process of the
feeder geometry. Computer Aided Manufacturing (CAM) is used as methodology that
involves numerical simulation of the casting process. Numerical simulation is used to
verify the validity of the optimized geometry of the feeding system.
Key Words: Sand Casting, Feeder System, Optimization, Genetic Algorithm,
Numerical Simulation
1. INTRODUCTION
Cooling of casting part causes shrinkage of material, therefore, the basic function of feeders
is to compensate for the deficiency of metal during casting part solidification. Unlike filling as a
relatively short process, feeding is a necessary, time-consuming and slow one. Casting part
cooling involves three phases of volume contraction (shrinkage) – liquid shrinkage,
solidification shrinkage and solid shrinkage [1]. Volume contraction is manifested through
some adverse phenomena, i.e. internal shrinkage voids, surface deformation or dishing, and
Received August 15, 2015 / Accepted January 28, 2016
Corresponding author: Nedeljko Duĉić
Faculty of Technical Sciences Ĉaĉak, University of Kragujevac, Svetog Save 65, 32000 Ĉaĉak
E-mail: nedeljko.ducic@ftn.kg.ac.rs
148
N. DUĈIĆ, Ţ. ĆOJBAŠIĆ, R. RADIŠA, R. SLAVKOVIĆ, I. MILIĆEVIĆ
surface puncture. The elimination of the above side effects is obtained by designing feeders
which are removed after cooling the casting part.
Optimal design of feeders, in addition to providing higher quality products, also
provides consumption savings of molten metal in the casting process. The total volume of
feeder should be minimized to improve the casting yield and productivity [2]. Jacob et al.
(2004) presented the integration of Genetic Algorithm (GA) and CAD in the design and
optimization of feeders in casting process. The synergy of CAD and genetic algorithms
generates a class of optimal feeders that also satisfy the standard criteria set up in literature
[3]. Tavakoli and Davami (2007) focus on the design of the feeding system in the sand
casting process using Topology optimization (Solid Isotropic Material with Penalisation
method) [4]. Tavakoli and Davami (2009) combine finite-difference analysis of the
solidification process with evolutionary topology optimization to systematically improve the
feeding system design [5]. Kotas et al. (2010) summarize the findings of multi-objective
optimization of a gravity sand-cast steel part for which an increase of the casting yield via
feeder optimization is considered. This is accomplished by coupling a casting simulation
software package with an optimization modulus [6]. Sutaria et al. (2011) present a new
approach to evaluating and optimizing casting feeding system design using feed-paths. The
feed-paths are computed by Vector Element Method (VEM) [7]. Choudhari et al. (2013)
describe the parameters to be considered while designing a feeder of an optimum size to get
higher casting Yield. Theoretical design model has been analyzed in ANSYS 12.0
simulation software to ensure that shrinkage porosity is completely eliminated from casting
[8]. Campbell (1991, 2004 & 2011) propose seven conditions for feeding casting part: 1. Do
not feed (unless necessary); 2. The heat-transfer requirement; 3. Mass-transfer (volume)
requirement; 4. The junction requirement; 5. Feed path requirement; 6. Pressure gradient
requirement and 7. Pressure requirement [9]. Those conditions were the starting points for
the design and optimization of feeders in the research studies.
This paper presents a methodology of designing and optimizing the feeders for casting a
cutting tooth holder. Based on the rules given in [9], using the Computer Aided Design/
Computer Aided Manufacturing systems and Genetic Algorithm (as a technique of the
artificial intelligence), the optimal feeding system for casting part is obtained. The paper
includes three crucial rules proposed by Campbell: (2) The heat-transfer requirement, (3)
Mass-transfer (volume) and (5) Feed path requirement. Those rules are included in the
optimization process through the initial setup of the feeding system, definition of fitness
function and definition constraints. Numerical simulation confirm the validity of the
implemented methodology for feeder design and optimization.
2. FEEDER SYSTEM DESIGN
Casting part is a part of excavator buckets, i.e. a cutting tooth holder (its CAD model
is shown in Fig. 1). Production technology of the cutting tooth holder is sand casting.
Molds for single use are made of a mixture of sand, binders and additives. A quartz sand
(SiO2) is usually used in making molds. The core is more exposed to the molten metal
and high temperatures. Therefore, the core is made of sand with rough grains.
CAD/CAM Design and the Genetic Optimization of Feeders for Sand Casting Process
149
Fig. 1 CAD models: cutting tooth, cutting tooth holder and an entire excavator bucket
Fig. 2 presents the casting part (a cutting tooth holder) and gating system with its marked
elements.
Fig. 2 Gating system for casting a cutting tooth holder
The first step in the feeding system design is to define the type of feeder, the number
of feeders and their position in the system of casting. The selected type of feeders is a
cylindrical open feeder because of the largest modulus (the cylinder has the smallest
surface area per volume). Place of feeders is determined so as to provide "directional
solidification", thus achieving the solidification of thinner to thicker cross sections of the
casting part and finally in the feeders. The number of feeders depends on the feeding
distance. There has been much experimental effort to determine feeding distances. The
early work by Pellini and his co-workers (summarized by Beeley (1972)) at the US Naval
Materials Laboratory is a classic investigation that has influenced the thinking on the
concept of feeding distance ever since. The number of feeders and feeding distance are
defined on the basis of the rules: “Feed path requirement - Feeding distance”. Therefore:
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N. DUĈIĆ, Ţ. ĆOJBAŠIĆ, R. RADIŠA, R. SLAVKOVIĆ, I. MILIĆEVIĆ
Ld 4.5T
(1)
where: Ld – feeding distance, T – thickness of the casting. Approximate dimensions of
casting part are 295x320x40 mm, so the recommended feeding distance Ld=180 mm.
Given the dimensions of the base of casting part (295x320 mm), it is necessary to use two
feeders positioned to meet the requirement of distance feeding Ld. Namely, the proposed
solution comprises two cylindrical symmetric feeders intended to compensate for volume
shrinkage of metal (Fig. 3).
Fig. 3 The proposed gating system with feeders
The next challenge in the design of the feeding system is the geometry feeders‟
optimization.
3. GA OPTIMIZATION OF FEEDER
Optimization of feeders is based on the use of Genetic Algorithm (GA) and rules:
(2) The heat-transfer requirement and (3) Mass-transfer (volume) requirement. Genetic
algorithms (GAs) are a family of adaptive search algorithms described and analyzed in
references [10-14]. GAs derive their name from the fact that they are loosely based on
models of genetic change in a population of individuals. These models consist of three
basic elements: (1) a Darwinian notion of „fitness‟, which governs the extent to which an
individual can influence future generations; (2) a „mating operator‟, which produces
offspring for the next generation; and (3) „genetic operators‟, which determine the genetic
makeup of offspring from the genetic material of the parents [13]. The main objective of the
optimization is to find the value of diameter (D) and height (H) of the feeder, with
minimizing volume of the feeder and respecting constraint specified on the rules.
CAD/CAM Design and the Genetic Optimization of Feeders for Sand Casting Process
151
Fig. 4 Algorithm of optimization and design of feeders
3.1. Fitness function
Defining the fitness function is one of the major steps of optimization via GA.
Creating the fitness function is based on the rule: The heat-transfer requirement. The
heat-transfer requirement for successful feeding can be stated as follows: the freezing time
of the feeder must be at least as long as the freezing time of the casting [9]. Casting
solidification time can be predicted using Chvorinov‟s Rule shown by the equation:
V
ts C
A
2
(2)
where t s is the total solidification time of the part or feeder, C is a mold constant, V is the
volume of metal, and A is the total surface area of the part or feeder. As the feeder serves
to compensate for the volume shrinkage in the casting part, it needs to be the final link of
the solidification chain, i.e. it should be the last to solidify in directional solidification
process. The longest solidification time for a given volume is the one where the shape of
152
N. DUĈIĆ, Ţ. ĆOJBAŠIĆ, R. RADIŠA, R. SLAVKOVIĆ, I. MILIĆEVIĆ
the part has the minimum surface area. The cylinder has the smallest surface area per
volume and can easily be made, which was crucial when designing the feeder. Feeder
modulus is taken to be a minimum of 20 % higher than the modulus of casting part, i.e.
modulus of part who feeds:
M feeder 1.2 M casting _ part
(3)
The modulus for a cylindrical feeder is given by the following equation:
M feeder
DH
2D 4H
(4)
Based on the values of volume and surface of the casting part, obtained by analyzing
CAD model (Vc=3661113.8 mm3 and Ac=253716.66 mm2), the casting part modulus is:
Mcasting_part = 14.43. As the feeders are symmetrical and designed that each of them should
feed half of the casting part, relation (5) gives the final expression of fitness function that
minimizes:
F M feeder 1.2
M casting _ part
2
DH
8.66 0
2D 4H
(5)
3.2. Defining constraints
Constraint is based on the rule Mass-transfer (volume) requirement. The fulfillment of
the conditions given in the rule “The heat-transfer requirement”, does not guarantee the
fulfillment of the conditions given in the rule “Mass-transfer (volume) requirement”.
Although the feeder may have been provided of such a size that it theoretically would
contain liquid until after the casting is solid, in fact it may still be too small to deliver the
volume of feed liquid that the casting demands. Thus it will be prematurely sucked dry,
and the resulting shrinkage porosity will extend into the casting. This is because they are
themselves freezing at the same time as the casting, depleting the liquid reserves of the
reservoir. Effectively, the feeder has to feed both itself and the casting. We can allow for
this in the following way. If we denote efficiency ε of the feeder as the ratio (volume of
available feed metal) / (volume of feeder, Vf) then the volume of feed metal is, of course
Vf. If the solidification shrinkage of the liquid is α, during freezing, then the feed
demand from both the feeder and casting together is α(Vf,Vc), and hence [15]:
V f (V f Vc )
(6)
Solving for the feeder volume yields:
Vf
Vc
V
D2 H
0.4 c
0.2Vc
2
4
(7)
where: α – solidification shrinkage of the liquid during freezing ( 4 , for steels the fcc
structure applies above 0.1% carbon where the melt solidifies to austenite), 14% –
for a normal cylindrical feeder, Vc/2 – because one feeder feeds half of the casting part.
The relation (7) is a constraint in the optimization process of geometry feeders.
CAD/CAM Design and the Genetic Optimization of Feeders for Sand Casting Process
153
3.3. Optimization results
Search domains of size values that are being optimized are given in Table 1. The
upper and lower limits are set in accordance with the proposed geometry of the casting
part and mold. Feeder height is limited by the mold height.
Table 1 Optimal values search domain
Lower bound
Upper bound
D (mm)
10
150
H (mm)
10
80
The population range is 100 individuals, with 100 generations at maximum. Stochastic
universal sampling algorithm is used in the selection. The population is seen as mapped on
the roulette-wheel, larger parts of the wheel belonging to strings with lower fitness (since it is
a minimization problem). N pointers are evenly placed on the roulette, N being the number
of individuals in a population. A population is generated by rotating the roulette once.
Uniform crossover is used as the operator of the crossing. The coefficient of the „crossover
fraction‟ is 0.8. Crossover fraction defines a portion of the new population derived from a
crossing (non-elite individuals), its value being between 0 and 1. No more than two elite
individuals are to be transferred to the next generation. The operation of GA is terminated
as early as the 92 generation due to the violation of specified limits. The obtained
optimized values are as follows: D=105 mm and H=80 mm. Fig. 5 shows the position of
the proposed feeders.
Fig. 5 The proposed gating system with feeders and sand mold
4. NUMERICAL SIMULATION
In addition to practical knowledge applied in foundries, simulation programs are used
to better understand the casting process and manage it more easily. The primary objective
of simulation is to confirm the validity of the methodology applied in the optimization and
design of the feeding system, and reduce the cost and ensure reliable and quality production
154
N. DUĈIĆ, Ţ. ĆOJBAŠIĆ, R. RADIŠA, R. SLAVKOVIĆ, I. MILIĆEVIĆ
accordingly. The entire casting process, from filling the mold cavity with liquid metal and
all the way to solidification and feeding is available as a good representation of the
physical model. Computer „Cold Casting‟ allows improving the process step by step [16].
Geometry casting is substantially facilitated, i.e. it is easy to perform changes in the geometry of
the casting part, the position of feeder and core, etc. as well as in the parameters of the casting
process (temperature of the casting part, filling speed, etc.). Modern software package
MagmaSoft 5 (Version 5.2) is used to simulate the casting process [17]. Model of casting
part, as well as models of the optimized gating system and feeders‟ geometry, are used in
the STL graphical format.
In our paper, the alloy which is being the subject of the casting simulation is not
available in the software database, therefore it is required that the data be loaded into the
database. A material with chemical composition and properties similar to the one to be
cast are chosen from the database. The chosen material has its name changed and loaded
into the user‟s part of the database whereupon it is possible to load a new composition of
chemical elements and features for the material to be cast. The properties of the new
material remain in the database. Chemical composition of an alloy affects its castability
and behavior during the casting process, as well as the properties of the final casting part.
The paper describes simulation of casting steel of chemical composition 0.5 % C, 1.4 %
Mn, 0.035 % P, 0.035 % S, 0.4 % Si, 0.1 % V. Compared to standard content of chemical
elements in steel (designation GS30Mn5), contents of carbon, manganese and vanadium
are changed. Initial temperatures of steel, sand mold and coldbox are 1600 °C, 40 °C and
20°C respectively. Basic limiting conditions at casting are as follows: Liquidus temperature
1498°C; Solidus temperature of 1406 °C; Criterion temperature 1: 1415.2 °C. At a
temperature named "criterion temperature 1" feeding of the cast part is calculated as the
default value; Criterion temperature 2: 1500 °C. Criterion temperature 2 is calculated
temperature based on the cooling rate and gradient of change in temperature over time;
Latent heat 254 kJ/kg; Feeding effectivity 30 %. Feeding effectivity criterion shows the
volume of material to which macroscopic feeding is possible; Surface tension coefficient
1.496 N/m. Subsequent to defining groups and types of materials in the casting system, it
is necessary to determine heat transfer coefficients – HTC for pairs of materials in
contact. In sand casting the mold and the core surfaces are coated so as to affect thermal
behavior of the mold and core.
Fig. 6 Heat transfer coefficient in Low Alloy (LA)-steel-coat pair
CAD/CAM Design and the Genetic Optimization of Feeders for Sand Casting Process
155
The database provides data on HTC coefficients for different material pairs. Fig. 6
shows the HTC for low-alloy steel and the mold coated with a layer of a particular
thickness. Mold cavity and core are coated with a layer of 0.1 mm thick. Heat transfer
coefficients – HTC applied in the simulation are used from the database software (in
Sand-Core we use constant HTC=800 W/m2K, in Feeder-Sand and Gate-Sand we use the
temperature-dependent Steel-Sand coefficient). Each HTC can be modified according to
the specific conditions in the drive, which includes the additional thermal conductivity.
Properly defined geometry network is in the basis of any simulation and is essential for
the accuracy of the simulation results. The fineness and density of the network enables
more accurate results but the simulation requires longer time. The total number of final
volumes is one million, whereby the number of volume elements pertaining to metal
157611, without critical elements present. To bring the simulation into correlation with
the physical process in reality requires that at least 3 elements of network volume are
provided between two adjacent walls. In our study, the number of volumes between two
adjacent walls on the axes x/y/z is 154/130/49, respectively. To define solidification, i.e.
cooling of the casting part, parameters and limiting conditions are required. Primarily,
time or temperature at which the solidification simulation ends need to be specified first.
In our study, the final temperature in the simulation is 200 °C.
The FEEDMOD criterion (Fig. 7) is specifically designed for sand casting. The objective
is to change the value of „Feedmod‟ to allow directional solidification to feeders. The highest
values should be in the feeder; they gradually become lower in the casting part. The values
of the thermal module confirm the validity of the proposed optimized feeders.
Fig. 7 Feedmod criterion
LIQUIDUS TO SOLIDUS criterion shows the time needed for a material to pass from
liquid to solid state. It helps us to discover areas in the casting part that solidify later,
particularly the volumes which solidified after the feeding time (isolated volume). Fig. 8
confirms the correctness of the designed gating system and feeders by this criterion, given
that the solidification period is the longest in the feeding area. HOT SPOT result shows the
volumes of metals that solidify last. It helps us to identify the areas containing residual
156
N. DUĈIĆ, Ţ. ĆOJBAŠIĆ, R. RADIŠA, R. SLAVKOVIĆ, I. MILIĆEVIĆ
liquid metal. It also enables us to foresee possible macroscopic feeding of the volume
from the feeder. Based on Fig. 9, there is a volume that solidifies in the feeder, which is a
prerequisite for a regular cast.
Fig. 8 Time elapsed from liquidus to solidus temperature
Fig. 9 HOT SPOT criterion
TOTAL POROSITY result shows cumulative porosity and microporosity in a single
result, i.e. maximum values of porosity and micro-porosity. Fig. 10 shows the X-ray total
porosity in the casting part and that through the mid-section of the feeder. Total porosity
is expressed through percentage (%). The x-ray view of the casting part renders conclusion that
alloy casting technology provides required quality.
CAD/CAM Design and the Genetic Optimization of Feeders for Sand Casting Process
157
Fig. 10 POROSITY criterion
5. CONCLUSION
The aim of this study is to present a methodology for optimizing the geometry of the
feeding system for sand casting process based on the application of genetic algorithm.
The result of the optimization are diameter and height of feeders (D=105 mm and H=80
mm), obtained as function of minimizing volume of feeder. Based on the obtained results
of the optimization, the entire feeding system is designed by means of CAD software.
Performed numerical simulations confirm the correctness of the designed optimized
feeding system in view of all the criteria. The proposed methodology of design and
optimization provides a complete filling of the mold cavity, directional solidification and
eliminate porosity.
Acknowledgements: The paper is a part of the research done within the project TR35037 and TR35015.
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3. Jacob, E., Sasikumar, R., Praveen, B., Gopalakrishna, V., 2004, Intelligent design of feeders for castings by
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8. Choudhari, C. M., Dalal, N. S., Ghude, A. P., Sankhe, P. P., Dhotre A.S., 2013, Design and analyisis of riser for
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12. De Jong, K., 1985, Genetic algorithms: A 10 year perspective, Proceedings of the First International Conference
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15. Kotas, P., 2011, Integrated modeling of process, structures and performance in cast parts, Ph.D. Thesis,
Technical University of Denmark - Department of Mechanical Engineering, Denmark, 50 p.
16. Fang, Y., Hu, J., Zhou, J., Yu, Y., 2014, Numerical Simulation of Filling and Solidification in Exhaust
Manifold Investment Casting, International Journal of Metalcasting, 8(4), pp. 39-45.
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English
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Environmental gut bacteria in European honey bees (Apis mellifera) from Australia and their relationship to the chalkbrood disease
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PloS one
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cc-by
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Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Funding: MN and MF were supported by grants
from the Rural Industry Research & Development
Corporation/Agrifutures (RIRDC grant numbers
HBE01-03 ANU58A and HBE 03-01 ANU58A) and
by the University of Canberra (internal funding). The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. Sheba Khan1, Doug Somerville2, Michael FreseID3, Murali Nayudu3* 1 Faculty of Health, University of Canberra, Canberra, Australia, 2 NSW Department of Primary Industries,
Goulburn, NSW, Australia, 3 Faculty of Science and Technology, University of Canberra, Canberra, Australia 1 Faculty of Health, University of Canberra, Canberra, Australia, 2 NSW Department of Primary Industries,
Goulburn, NSW, Australia, 3 Faculty of Science and Technology, University of Canberra, Canberra, Australia * Murali.Nayudu@canberra.edu.au * Murali.Nayudu@canberra.edu.au a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Abstract We report on aerobic “environmental” bacteria isolated from European honey bees (Apis
mellifera). We determined the number of culturable aerobic bacteria in the gut of nurse bees
sampled from locations around Australia. Bees from healthy colonies had 107–108 aerobic
bacteria per g of bee gut, while bees from colonies with chalkbrood consistently had signifi-
cantly fewer bacteria (104–105 bacteria per g). When colonies recovered from chalkbrood,
bacterial numbers returned to normal levels, suggesting that counting aerobic bacteria in
the gut could be used to predict an outbreak of the disease. Furthermore, Western Austra-
lian bees from the “Better Bees” program (bred to promote hygienic behaviour) had signifi-
cantly higher numbers of aerobic gut bacteria compared to regular bees from healthy
colonies. Bacteria with the ability to inhibit the chalkbrood pathogen were found in most
bees from regular colonies (> 60%) but only in a few “Better Bees” (10%). Phylogenetic anal-
ysis of aerobic bacterial isolates that inhibited the chalkbrood pathogen revealed a close
relationship (>97% sequence identity) to the genera Bacillus, Klebsiella, Pantoea, Hafnia,
and Enterobacter (bacteria that have previously been isolated from honey bees), but we
also isolated Maccrococcus and Frigoribacterium species (bacteria that were not previously
identified in bees). Finally, we investigated the ability of bacteria to inhibit the chalkbrood
fungus Ascosphaera apis. Mass spectroscopy analysis revealed that the bee gut isolates
Frigoribacterium sp. and Bacillus senegalensis produce gluconic acid. We further found that
this simple sugar is involved in chalkbrood fungal hyphal lysis and cytoplasmic leakage. Our
findings suggest that “environmental” gut bacteria may help bees to control the chalkbrood
pathogen. PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Khan S, Somerville D, Frese M, Nayudu M
(2020) Environmental gut bacteria in European
honey bees (Apis mellifera) from Australia and their
relationship to the chalkbrood disease. PLoS ONE
15(8): e0238252. https://doi.org/10.1371/journal. pone.0238252 Editor: Wolfgang Blenau, Universitat Leipzig,
GERMANY Editor: Wolfgang Blenau, Universitat Leipzig,
GERMANY Received: April 3, 2020
Accepted: August 12, 2020
Published: August 28, 2020 Copyright: © 2020 Khan et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Environmental gut bacteria in European
honey bees (Apis mellifera) from Australia and
their relationship to the chalkbrood disease Sheba Khan1, Doug Somerville2, Michael FreseID3, Murali Nayudu3* PLOS ONE PLOS ONE Introduction European honey bees (Apis mellifera) were first introduced to Australia in 1822 for honey pro-
duction and soon became widespread. Bees are not only key pollinators of many agricultural
crops but also became important pollinators of some native Australian plant species [1]. The
production of honey and honey-derived products directly contributes 101 million AUD per PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 1 / 22 PLOS ONE Bee gut bacteria and chalkbrood Competing interests: AgriFutures (formerly known
as Rural Industry Research & Development
Corporation) is an Australian government body that
funds basic and applied research. Therefore,
AgriFutures should not be considered a
“commercial funder”. We confirm that the
involvement of AgriFutures “does not alter our
adherence to PLOS ONE policies on sharing data
and materials” annum to the Australian economy. More importantly, honey bees provide pollination to a
wide range of agricultural and horticultural crops with an estimated benefit to the economy of
about 2 billion AUD per annum [2]. In Australia, European honey bees have very different
pollen and nectar sources, compared with other continents [3]. The unique flora that has
evolved on the Australian “island” continent since the breakup of the Gondwana superconti-
nent in the Jurassic period [4] enables the production of some unique honeys, such as Leather-
wood honey and Leptospermum (Manuka) honey [5]. Furthermore, Australia is still free of the
Varroa mite [6] and colony collapse disorder (CCD)—in contrast to other parts of the world
[7]. Chalkbrood is a disease of the honey bee brood that is caused by the heterothallic fungus
Ascosphaera apis [8]. In Australia, the chalkbrood disease was first detected in 1993; it quickly
became endemic [9] and is now the most important disease in the local bee industry [10]. Physical, chemical, and biological stress factors predispose colonies to chalkbrood, such as
extreme (high and low) temperatures, high humidity, environmental pollution, pesticide poi-
soning, parasite infestations, and predator attacks [11]. The severity of a chalkbrood outbreak
depends on the pathogenicity of the fungus, the vitality of the colony (e.g., hygienic behavior),
and specific environmental factors [12]. Some measures can be taken to reduce the probability
of disease outbreaks, such as maintaining colonies with sufficient numbers of bees, not allow-
ing bees to winter in brood chambers that are too large [13], or re-queening the colony with a
queen from a more resistant stock [14]. Competing interests: AgriFutures (formerly known
as Rural Industry Research & Development
Corporation) is an Australian government body that
funds basic and applied research. Therefore,
AgriFutures should not be considered a
“commercial funder”. We confirm that the
involvement of AgriFutures “does not alter our
adherence to PLOS ONE policies on sharing data
and materials” Introduction Enlarging hive entrances to aid ventilation and reduce
moisture accumulation may also help [13]. In a chalkbrood-infested colony, each infected
larva can produce up to 108–109 spores [8]. Chalkbrood mummies are frequently ejected from
the colony by nurse bees, which reduces the pathogen load in the hive. Thus, the hygienic
behavior of nurse bees is a key factor in halting the spread of the disease and in promoting
recovery [15]. A wide range of microorganisms were found to be associated with honey bees and their
food (i.e., nectar, pollen, honey, beebread, and propolis [16]). The gut microbiome of bees and
other insects has been studied in great detail. For example, microorganisms digest and ferment
plant cell wall components [17], produce essential vitamins [18], and prevent harmful patho-
gens from colonizing the gut through the occupation of key niches [18] and/or the production
of metabolites that prime and/or enhance host immune responses [17]. Furthermore, it has
been postulated that bee gut bacteria contribute to general hygiene, pathogen inhibition, and
bee bread preservation [19, 20]. Molecular analysis of honey bee gut microbial communities
revealed eight characteristic, strictly anaerobic or microaerophilic phylotypes that were found
across several environments and geographical locations [21–24]. The most frequently found
“core” bacteria of adult honey bees are Gram-negative Proteobacteria (e.g., Snodgrassella and
Gilliamella ssp. [25]) and Gram-positive Firmicutes (e.g., Lactobacillus and Lactobacillus ssp. [26]). Less frequently found are Bifidobacterium ssp. [27], Frischella, Bartonella, Parasacchari-
bacter, and Gluconobacter-related species [26]. Bees acquire these bacteria early in life and
most retain them throughout their lifetime [28]. Martha Gilliam first showed that the bee gut
also contains a diverse range of aerobic bacterial species and that some of these bacteria (e.g.,
Bacillus ssp.) inhibit the chalkbrood pathogen [20]. These “environmental” bacteria may only
colonize the bee gut opportunistically but nevertheles may have important roles to play in the
digestion of food or the control of pathogens. This study reports on aerobic bacteria from the gut of European honey bees from Australia. We chose to investigate nurse bees, because they rarely leave the hive, look after the brood, and
are responsible for feeding the larvae, which means that it is the gut bacteria of nurse bees that
are most likely passed on to the next generation of bees [29, 30]. Furthermore, nurse bees
remove infected chalkbrood larvae from the hive [15]. Materials and methods The main steps involved in the isolation and characterization of aerobic gut bacteria from Aus-
tralian honey bees are summarized in a flowchart (S1 Fig). Honey bee sampling and processing of the bee gut Samples were collected from the major honey producing regions across Australia during the
Australian late spring, summer, and early autumn (when bees are most active). We sampled
from the Australian Capital Territory (ACT; 7 hives), New South Wales (NSW; 29 hives),
Queensland (QLD; 10 hives), Victoria (VIC; 10 hives), Tasmania (TAS; 10 hives), South Aus-
tralia (SA; 6 hives), and Western Australia (WA; 20 hives). Each hive was deconstructed for
sampling, assessed for the presence of chalkbrood, and bees taken from the brood area, which
made it highly likely that most sampled bees were indeed nurse bees. Most bees were trans-
ported to the laboratory on the same day where they were kept at room temperature until they
were killed for the isolation of aerobic bacteria from the bee gut. If bees were shipped by mail
(over-night), they were sent in queen mailing cages plugged with irradiated sugar candy. For each honey bee colony, bacteria from four randomly selected nurse bees were analyzed. Bees were placed in a fridge at 4˚C for a minimum of three but not more than four hours. The
bees were carefully removed from the fridge, the sternum of each bee was crushed using sterile
tweezers, and a second pair of sterile tweezers was used to pull out the entire gastrointestinal
tract (i.e., ventriculus, ileum, and rectum). The crop was cut off, the remaining gut was trans-
ferred into an Eppendorf tube with 1 ml of sterile dilute nutrient broth (Sigma-Aldrich; cata-
logue number, 03856), and the fresh weight was determined (to three decimal places). Introduction Thus, nurse bees are key players in both PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 2 / 22 PLOS ONE Bee gut bacteria and chalkbrood the spread and the recovery from chalkbrood [15, 29, 30]. By comparing nurse bees from
healthy colonies with those from chalkbrood-infected colonies in the same apiaries, we tested
the hypothesis that changes to the number of culturable (aerobic) gut bacteria correlate with
the appearance (or disappearance) of the chalkbrood disease. Furthermore, using 16S DNA
sequencing, we determined the identity of selected aerobic bee gut bacteria that were found to
inhibit the fungal pathogen Ascosphaera apis, and we investigated the mechanism by which
bacteria inhibit the growth of A. apis. Chalkbrood inhibition assay All bacterial isolates were tested for their ability to inhibit the chalkbrood and/or the “take-all”
fungus using previously described standard bioassays [20]. Briefly, plugs of chalkbrood were
placed between the middle and the rim of PDA plates before the plates were streaked with bac-
teria on the opposite side. Control plates had the fungal plug placed in the middle of the plate. The plates were then incubated for 10–14 days at 18˚C and fungal inhibition was determined
with an Alpha Digidoc image system (Sigma Aldrich) that quantifies the plate surface area
with fungal growth. Results are expressed as mean % inhibition, using the following formula
[34]: % inhibition = [1- (fungal growth/control growth)] × 100. Isolation of bacteria Bacteria were isolated on TSA (Oxoid; catalogue number, CM0131), a general purpose
medium; eosin methylene blue agar (EMB; Oxoid; catalogue number, CM0069) for enteric
bacteria; modified Gould medium (mS1) for the selective isolation of Pseudomonas sp.; and
glucose-calcium carbonate medium (G-CaCO3) for the selective isolation of Gluconobacter
ssp. [31]. Potato dextrose agar (PDA; Oxoid; catalogue number, CMO139) was used for fungus
inhibition assays and the isolation of bacterial hydrophilic compounds. For light microscopy
studies, thinly plated PDA plates were poured that contained a lower potato dextrose concen-
tration than normal (i.e., 10 g/liter). The pH indicator bromocresol purple (5,5’-dibromo-o-
cresolsulfonphthalein) was added to PDA to detect the production of acidic bacterial metabo-
lites. The indicator was added to the medium (15 mg/liter) before autoclaving and the pH was
adjusted through the dropwise addition of 5 M NaOH until the medium was deep purple in
color (pH 6.8); a color change to yellow (pH < 5) indicated acidification. Bacteria were
streaked on media and incubated at 25˚C for 2 days. Potato dextrose broth (PDB; Fisher Scien-
tific; catalogue number, BD 254920) was used to grow bacteria for preparing crude extracts of
the Pseudomonas strain AN5 and the AN5 transposon mutant derivatives AN5MN1 and
AN5MN2. Pseudomonas strain AN5 produces gluconic acid (approximately 0.5 mg/ml) on
PDA medium, while the two independent transposon mutants AN5MN1 and AN5MN2 do
not produce any gluconic acid [33]. Gram staining Bacteria were stained using a Gram staining kit (Sigma-Fluka; catalogue number 77730) and
observed under a light microscope (at a magnification of 1,000×). Quantification of bacteria The bee gut was crushed using sterile sticks and vortexed for 1 min. Serial dilutions of the
resulting suspension were prepared in nutrient broth and 200 μl of each dilution along with
the neat (both in duplicates) was spread over the surface of agar plates with a sterile spreader. The plates were incubated at 25˚C for 2 days. Bacterial counts were determined on tryptic soy
agar (TSA [31]) on dilution plates that had countable numbers of bacteria (i.e., 20–200 colo-
nies per plate). We calculated the number of bacteria per g bee rather than per bee to allow
meaningful comparisons of bee gut bacterial numbers across colonies and apiaries from differ-
ent environments/geographical locations. All bacterial counts (CFU/g) were log10-transformed and evaluated statistically using Gen-
stat, version 9.0 [32]. The Mann-Whitney U (Wilcoxon rank-sum) test for non-parametric
analysis was used to compare different samples. Observed differences were considered signifi-
cant at P < 0.05. In conjunction with the Mann-Whitney U test, a one-way analysis of variance
(ANOVA) was carried out to compare the means and the least significant difference between
sample groups. 3 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 PLOS ONE Bee gut bacteria and chalkbrood PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 PLOS ONE Cluster
Bacterial isolate
Origin of isolate
Bacillus group
16H10Pw4.9
Bermagui (NSW)
16H13Tw3.12
Bermagui (NSW)
17H2Gw4.15
Launceston (TAS)
17H3Gw3.4
Launceston (TAS)
21H1Gw3.10
Perth (WA)
23H1Tw4.20
Cooke Plains (SA)
24H1Tw3.19
Cooke Plains (SA)
30H1Tw2.10
Tubbut (VIC)
30H2Gw2.10
Tubbut (VIC)
30H2Gw2.12
Tubbut (VIC)
30H2Gw3.18
Tubbut (VIC)
30H2Gw3.5
Tubbut (VIC)
ET6
Canberra (ACT)
Enteric group
14H12Pw3.4
Goulburn (NSW)
16H13Pw2.4
Bermagui (NSW)
16H14Ew3.3
Bermagui (NSW)
20H2Ew2.1
Perth (WA)
Macrococcus group
ET2
Canberra (ACT)
Frigoribacterium group
DT4
Canberra (ACT)
https://doi.org/10.1371/journal.pone.0238252.t001
PLOS ONE
Bee gut bacteria and chalkbrood Table 1. Classification, name and origin of aerobic bacteria isolated from the gut of Australian honey (nurse) bees. Cluster
Bacterial isolate
Origin of isolate
Bacillus group
16H10Pw4.9
Bermagui (NSW)
16H13Tw3.12
Bermagui (NSW)
17H2Gw4.15
Launceston (TAS)
17H3Gw3.4
Launceston (TAS)
21H1Gw3.10
Perth (WA)
23H1Tw4.20
Cooke Plains (SA)
24H1Tw3.19
Cooke Plains (SA)
30H1Tw2.10
Tubbut (VIC)
30H2Gw2.10
Tubbut (VIC)
30H2Gw2.12
Tubbut (VIC)
30H2Gw3.18
Tubbut (VIC)
30H2Gw3.5
Tubbut (VIC)
ET6
Canberra (ACT)
Enteric group
14H12Pw3.4
Goulburn (NSW)
16H13Pw2.4
Bermagui (NSW)
16H14Ew3.3
Bermagui (NSW)
20H2Ew2.1
Perth (WA)
Macrococcus group
ET2
Canberra (ACT)
Frigoribacterium group
DT4
Canberra (ACT)
https://doi.org/10.1371/journal.pone.0238252.t001 identify 16S rRNA gene sequences with greater than 96% identity and the highest level of simi-
larity, as estimated using expect values [36]. For sequence comparisons, we used only those
16S rRNA gene sequence in the NCBI data base that were derived from known characterized
bacterial strains. Where possible, sequences from strains of the American Type Culture Collec-
tion (ATCC) were used for creating the phylogenetic tree. Partial 16S rRNA gene sequences
were determined for 19 bacterial isolates (Table 1). identify 16S rRNA gene sequences with greater than 96% identity and the highest level of simi-
larity, as estimated using expect values [36]. For sequence comparisons, we used only those
16S rRNA gene sequence in the NCBI data base that were derived from known characterized
bacterial strains. Where possible, sequences from strains of the American Type Culture Collec-
tion (ATCC) were used for creating the phylogenetic tree. Partial 16S rRNA gene sequences
were determined for 19 bacterial isolates (Table 1). 16S rDNA sequences from bacterial isolates and known GenBank sequences were grouped
using similarity scores and knowledge of their taxonomy. Separate datasets were compiled for
each major grouping. Out group sequences from other bacterial genera, including a representa-
tive from each of the groups identified in this work, were included in each sequence dataset. PLOS ONE Sequence datasets were aligned using the Fast Fourier Transform (MAFFT) alignment program
[37], a program that is frequently used to align large numbers of sequences for phylogenetic
analysis [38]. Maximum likelihood phylogenetic trees were generated by using PhyML 3.0 and
sub-tree pruning and regrafting from 10 random starting trees [39]. Each of the substitution
models available in PhyML were used, including the most complex model, a general time-
reversible model with a proportion of invariant sites, and a gamma distribution of site-rate vari-
ants across categories: GTR+I+G [40]. Bootstrap values > 50% are shown at the nodal branches. Corresponding phylogenetic trees were derived from the partial 16S rRNA gene sequences. DNA isolation and sequencing Total genomic DNA was isolated from selected bacterial strains using the DNeasy Blood & Tis-
sue Kit (Qiagen; catalogue number, 69504). Multiplex PCR was used to amplify 16S rRNA
gene fragments using the Multiplex PCR Kit (Qiagen; catalogue number, 206143). 16S rRNA
amplification was carried out with the two primers: primer BSF 8/20 (5’-AGAGTTTGATCCT
GGCTCAG-3’) and primer BSR 534/18 (5’-ATTACCGCGGCTGCTGGC-3’). These primers
were designed using sequence information from the European Ribosomal RNA website [35]. Agarose gel electrophoresis was used to visualize the amplified 500-bp 16S rRNA DNA frag-
ment. Bands with the correct size were excised from gels and purified using the Qiagen QIA
quick Gel Extraction Kit. DNA sequencing of this fragment was performed at the Australian
Genome Research Facility (AGRF) at the University of Queensland, Brisbane. Partial 16S rRNA gene sequences were corrected and trimmed (as needed) using Sequencer
6.0 before they were subjected to a BLAST search of the GenBank non-redundant database to PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 4 / 22 PLOS ONE Bee gut bacteria and chalkbrood PLOS ONE identify 16S rRNA gene sequences with greater than 96% identity and the highest level of simi-
larity, as estimated using expect values [36]. For sequence comparisons, we used only those
16S rRNA gene sequence in the NCBI data base that were derived from known characterized
bacterial strains. Where possible, sequences from strains of the American Type Culture Collec-
tion (ATCC) were used for creating the phylogenetic tree. Partial 16S rRNA gene sequences
were determined for 19 bacterial isolates (Table 1). 16S rDNA sequences from bacterial isolates and known GenBank sequences were grouped
using similarity scores and knowledge of their taxonomy. Separate datasets were compiled for
each major grouping. Out group sequences from other bacterial genera, including a representa-
tive from each of the groups identified in this work, were included in each sequence dataset. Sequence datasets were aligned using the Fast Fourier Transform (MAFFT) alignment program
[37], a program that is frequently used to align large numbers of sequences for phylogenetic
analysis [38]. Maximum likelihood phylogenetic trees were generated by using PhyML 3.0 and
sub-tree pruning and regrafting from 10 random starting trees [39]. Each of the substitution
models available in PhyML were used, including the most complex model, a general time-
reversible model with a proportion of invariant sites, and a gamma distribution of site-rate vari-
ants across categories: GTR+I+G [40]. Bootstrap values > 50% are shown at the nodal branches. Corresponding phylogenetic trees were derived from the partial 16S rRNA gene sequences. Anti-fungal compound extraction and detection
The aerobic ET2 bee gut bacterial isolate was spread onto five PDA plates and incubated at
25˚C for five days before the contents of the plates were cut into cubes and transferred to a
250-ml flask with 100 ml of a water/isopropanol solution (40:60). The flask was plugged with
bungs and placed on a shaker for 1 h, before the contents were strained and centrifuged at
6,000 rpm for 15 min. The pellet (agar) was discarded and the supernatant was freeze-dried,
resuspended in 100 ml of acetone, transferred to a new glass beaker, and refrigerated at 4˚C
Table 1. Classification, name and origin of aerobic bacteria isolated from the gut of Australian honey (nurse) bees. Anti-fungal compound extraction and detection The aerobic ET2 bee gut bacterial isolate was spread onto five PDA plates and incubated at
25˚C for five days before the contents of the plates were cut into cubes and transferred to a
250-ml flask with 100 ml of a water/isopropanol solution (40:60). The flask was plugged with
bungs and placed on a shaker for 1 h, before the contents were strained and centrifuged at
6,000 rpm for 15 min. The pellet (agar) was discarded and the supernatant was freeze-dried,
resuspended in 100 ml of acetone, transferred to a new glass beaker, and refrigerated at 4˚C PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 5 / 22 PLOS ONE Bee gut bacteria and chalkbrood overnight. After another centrifugation step (6,000 rpm for 10 min), the sample was trans-
ferred to a new glass beaker and left uncovered in a laminar flow hood until all of the acetone
had evaporated [33]. Finally, the sample was resuspended in 5 ml of sterile water. All isolations
were performed in duplicate. A 1-ml aliquot of the ET2 hydrophilic extract was tested for the presence of gluconic acid in
a VG QUATTRO II mass spectrometer [33]. Mass spectrometry of pure gluconic acid (Merck;
catalogue number, Sigma-Aldrich G1951) was carried out as a control. Methanol was used
between samples to purge compound traces from the mass spectrometer. A collision-induced
dissociation (CID) analysis of the putative gluconic acid peak was also performed to confirm
the presence of gluconic acid polymers on any gluconic acid peaks observed. Crude extracts of 2-day PDB cultures of the Pseudomonas strain AN5, or that of the inde-
pendent transposon mutants AN5MN1 and AN5MN2, were obtained by filtration. The filtrate
was then freeze-dried to concentrate any metabolites and resuspended in sterile water for use
in bioassays [33]. All tests were performed as duplicates. Briefly, a plug of the chalkbrood fun-
gus was placed in the middle of a thinly poured agar plate, sterile paper discs were placed sym-
metrically in each quadrant, and 100 μl of either water (as control) or crude bacterial extracts
were pipetted onto each disc. The plates were then incubated at 18˚C for 8 days and the effect
of the various treatments were examined by direct observation under a light microscope (at a
magnification of 400×). PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 Nurse bees from healthy colonies have more aerobic gut bacteria than
nurse bees from chalkbrood-infected colonies For this study, 98 bee colonies were sampled from 33 different locations across Australia, cov-
ering the major honey production areas in all states and territories except the Northern Terri-
tory (Fig 1). When chalkbrood-infected colonies were sampled, healthy colonies from the
same apiary were also sampled. From each colony that was selected for sampling, nurse bees
were removed from the brood area, taken to the laboratory, and processed for the isolation of
bee gut bacteria. Only the mid- to hindgut was harvested for bacterial isolation. The weight of
the bee gut varied considerably (i.e., between 10 and 50 mg). Therefore, we calculated the
number of bacteria per gram of bee gut to better compare bee gut bacterial numbers across dif-
ferent colonies and apiaries. Of note, we did not attempt to cultivate the anaerobic or micro-
aerophilic “core” bacteria of the bee gut, our methods were chosen to analyze the abundance
of aerobic “environmental” gut bacteria. All bees from healthy colonies (47 hives, 4 bees per hive, i.e., n = 47) had high numbers of
aerobic gut bacteria (107–108 CFU/g) compared with bees from chalkbrood-infected colonies
(31 hives, 4 bees per hive, i.e., n = 31), which had significantly (P < 0.001) fewer bacteria (104–
105 CFU/g; Fig 2). We also investigated changes to the number of aerobic gut bacteria in chalkbrood-infected
hives over time in an apiary near the township of Goulburn (New South Wales) that had
numerous chalkbrood-infected hives. From that apiary, bees from 8 colonies were sampled
when the colonies showed signs of the chalkbrood disease. The same hives were sampled again
21 weeks later when the colonies had recovered, and the signs of the disease were no longer
observed. For comparisons, bees from healthy colonies in the same apiary were also sampled
(Fig 3). The results confirm our earlier findings that bees from chalkbrood-infected colonies
contained fewer bacteria than bees from colonies that did not display any signs of the disease. Interestingly, the disappearance of the chalkbrood disease largely coincided with a recovery in
bacterial numbers, even if aerobic bacterial numbers in the gut of bees from colonies that had PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 6 / 22 PLOS ONE Bee gut bacteria and chalkbrood Fig 1. Origin of samples. Australian states and territories from which nurse bees were collected for this study. Nurse bees from healthy colonies have more aerobic gut bacteria than
nurse bees from chalkbrood-infected colonies Red
dots indicate the location of apiaries sampled in this study. NSW, New South Wales; VIC, Victoria; WA, Western
Australia; QLD, Queensland; SA, South Australia; TAS, Tasmania; ACT, Australian Capital Territory. https://doi.org/10.1371/journal.pone.0238252.g001 Fig 1. Origin of samples. Australian states and territories from which nurse bees were collected for this study. Red
dots indicate the location of apiaries sampled in this study. NSW, New South Wales; VIC, Victoria; WA, Western
Australia; QLD, Queensland; SA, South Australia; TAS, Tasmania; ACT, Australian Capital Territory. https://doi.org/10.1371/journal.pone.0238252.g001 https://doi.org/10.1371/journal.pone.0238252.g001 just recovered from chalkbrood were slightly lower compared with bees from healthy colonies
without a (recent) history of chalkbrood. The results inspired us to analyze bees from the Western Australian “Better Bees” program. The “Better Bees” were bred for enhanced activity and improved hygienic behavior [41]. We
compared aerobic bacterial bee gut counts from 10 healthy “Better Bee” colonies in Western
Australia with normal bee colonies from the same area. The results show that bees from the
“Better Bees” program had significantly more aerobic gut bacteria than bees from regular bee
colonies (i.e.,108.5–109 bacteria vs 107–108 bacteria per g, respectively; P = 0.002; Fig 4). PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 Bees from “Better Bee” colonies have fewer aerobic chalkbrood inhibiting
gut bacteria than normal colonies Error bars represent the SEM with a 5% least significant difference (LSD) level of 1.095. https://doi.org/10.1371/journal.pone.0238252.g003 https://doi.org/10.1371/journal.pone.0238252.g003 https://doi.org/10.1371/journal.pone.0238252.g003 based on the presence of chalkbrood-inhibiting bacteria. There was no obvious correlation
between the geographical location of an apiary and the number of chalkbrood-inhibiting gut
bacteria. However, more apiaries need to be tested to confirm this result. Bees from “Better Bee” colonies have fewer aerobic chalkbrood inhibiting
gut bacteria than normal colonies Of all healthy colonies that were tested, 61% had bees containing aerobic gut bacteria that
inhibited the chalkbrood pathogen. Interestingly, 73% of colonies tested with chalkbrood
symptoms had bees with aerobic gut bacteria that inhibited the chalkboard pathogen. The per-
centage increased even further to 83% for colonies that had recently recovered from chalk-
brood. Somewhat unexpectedly, only 10% of the “Better Bee” colonies had bees with
chalkbrood-inhibiting bacteria, which is significantly below that of other apiaries from West-
ern Australia. This suggests that the “resistance” to chalkbrood in the “Better Bee” lines is not PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 7 / 22 PLOS ONE Bee gut bacteria and chalkbrood Fig 2. Aerobic gut bacterial numbers (CFU/g) in nurse bees from healthy hives (n = 47) and chalkbrood-infected
hives (n = 31). Bees were collected from around Australia. The difference between the two groups is statistically
significant (P < 0.001). Error bars represent the standard error of the mean (SEM) with a 5% least significant
difference (LSD) level of 0.716. https://doi.org/10.1371/journal.pone.0238252.g002
ONE
Bee gut bacteria and chalkbrood Fig 2. Aerobic gut bacterial numbers (CFU/g) in nurse bees from healthy hives (n = 47) and chalkbrood-infected
hives (n = 31). Bees were collected from around Australia. The difference between the two groups is statistically
significant (P < 0.001). Error bars represent the standard error of the mean (SEM) with a 5% least significant
difference (LSD) level of 0.716. https://doi.org/10.1371/journal.pone.0238252.g002 8 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 PLOS ONE Bee gut bacteria and chalkbrood Fig 3. Aerobic gut bacterial numbers (CFU/g) in nurse bees from healthy hives (n = 10), chalkbrood-infected hives
(n = 10), and chalkbrood-infected hives 21 weeks after the disappearance of disease signs (recovered; n = 10). All
bees were collected from a single apiary in New South Wales. The differences between all groups are statistically
significant (P < 0.001). Error bars represent the SEM with a 5% least significant difference (LSD) level of 1.095. https://doi org/10 1371/journal pone 0238252 g003 Fig 3. Aerobic gut bacterial numbers (CFU/g) in nurse bees from healthy hives (n = 10), chalkbrood-infected hives
(n = 10), and chalkbrood-infected hives 21 weeks after the disappearance of disease signs (recovered; n = 10). All
bees were collected from a single apiary in New South Wales. The differences between all groups are statistically
significant (P < 0.001). Characterization of aerobic bee gut bacteria that inhibit the chalkboard
pathogen To determine bee gut bacterial numbers, bacteria were usually grown on TSA media, but
we also inoculated EMB, mS1 Gould, and glucose-CaCO3 agar to isolate bacteria that do not
grow readily on TSA agar (S2 Fig). Gram staining was carried out for all bacterial isolates
obtained in this study. We found that, throughout Australia, more Gram-negative than
Gram-positive bacteria were observed in the gut of healthy bees (Fig 5), which is in line with
earlier observations by Gilliam [20]. We also isolated a small number of Gram-variable bacte-
ria (Fig 5). PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 9 / 22 Of the 1,758 aerobic bacterial isolates from the gut of healthy bees, only 170 strongly inhib-
ited the chalkbrood fungus (% inhibition, 30%; for examples without and with anti-fungal
activity, see Fig 6A and 6B, respectively). A genetic analysis using randomly amplified
Fig 4. Aerobic gut bacterial numbers (CFU/g) in healthy nurse bees from “Better Bee” colonies (n = 10) and bees
from regular breeding colonies (n = 10). All bees were collected from apiaries in Western Australia. The difference
between the two groups is statistically significant (P = 0.002). Error bars represent the SEM with a 5% least significant
difference (LSD) level of 0.562. https://doi.org/10.1371/journal.pone.0238252.g004
Bee gut bacteria and chalkbrood PLOS ONE Bee gut bacteria and chalkbrood Fig 4. Aerobic gut bacterial numbers (CFU/g) in healthy nurse bees from “Better Bee” colonies (n = 10) and bees
from regular breeding colonies (n = 10). All bees were collected from apiaries in Western Australia. The difference
between the two groups is statistically significant (P = 0.002). Error bars represent the SEM with a 5% least significant
difference (LSD) level of 0.562. Fig 4. Aerobic gut bacterial numbers (CFU/g) in healthy nurse bees from “Better Bee” colonies (n = 10) and bees
from regular breeding colonies (n = 10). All bees were collected from apiaries in Western Australia. The difference
between the two groups is statistically significant (P = 0.002). Error bars represent the SEM with a 5% least significant
difference (LSD) level of 0.562. https://doi.org/10.1371/journal.pone.0238252.g004 https://doi.org/10.1371/journal.pone.0238252.g004 Of the 1,758 aerobic bacterial isolates from the gut of healthy bees, only 170 strongly inhib-
ited the chalkbrood fungus (% inhibition, 30%; for examples without and with anti-fungal
activity, see Fig 6A and 6B, respectively). Characterization of aerobic bee gut bacteria that inhibit the chalkboard
pathogen A genetic analysis using randomly amplified 10 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 PLOS ONE Bee gut bacteria and chalkbrood Fig 5. Gram staining properties of aerobic gut bacteria isolated from European nurse honey bees from in
Australia. Isolates were analyzed from New South Wales (NSW), Victoria (VIC), Tasmania (TAS), South Australia
(SA), Western Australia (WA), and Queensland (QLD). https://doi.org/10.1371/journal.pone.0238252.g005 Fig 5. Gram staining properties of aerobic gut bacteria isolated from European nurse honey bees from in
Australia. Isolates were analyzed from New South Wales (NSW), Victoria (VIC), Tasmania (TAS), South Australia
(SA), Western Australia (WA), and Queensland (QLD). Fig 5. Gram staining properties of aerobic gut bacteria isolated from European nurse honey bees from in
Australia. Isolates were analyzed from New South Wales (NSW), Victoria (VIC), Tasmania (TAS), South Australia
(SA), Western Australia (WA), and Queensland (QLD). https://doi.org/10.1371/journal.pone.0238252.g005 https://doi.org/10.1371/journal.pone.0238252.g005 https://doi.org/10.1371/journal.pone.0238252.g005 https://doi.org/10.1371/journal.pone.0238252.g005 polymorphic DNA revealed that 158 of the 170 isolates are not closely related. Next, we investi-
gated the 158 aerobic bee gut isolates that were different from each other and that strongly
inhibited the chalkbrood pathogen. The nature of metabolites produced by selected bacterial
isolates was determined using PDA plates supplemented with the pH indicator bromocresol
purple. Results indicate that aerobic bee gut bacteria produced a range of acidic to alkaline
metabolites (as exemplified in S3 Fig). PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 Gluconic acid production by bacteria and its effect on the chalkbrood
pathogen By growing bacteria on PDA plates, we found that one of our chalkbrood-inhibiting aerobic
bacterial bee gut isolates, ET2, produced acidic metabolites when glucose, galactose, or man-
nose were provided as the sole carbon source (S3 Fig). This metabolic activity is indicative of
the presence of a glucose oxidase-like enzyme [42]. The production of gluconic acid is of inter-
est, because this simple sugar is involved in the inhibition of the “take-all” wheat pathogen
Gaeumannomyces graminis var. tritici (Ggt) [33]. Thus, it was not surprising to find that ET2
also strongly inhibited the “take-all” pathogen in a PDA plate bioassay (% inhibition, 30%;
similar to the inhibition of chalkbrood by the Pseudomonas strain AN5; Fig 6C). This observa-
tion suggests that ET2 produces gluconic acid, and that gluconic acid contributes to the inhibi-
tion of the chalkbrood pathogen. As gluconic acid is a water-soluble compound, we extracted
hydrophilic metabolites from ET2-inoculated PDA plates and analyzed the extracts using mass
spectroscopy. The analysis revealed a peak at approximately 195 m/z that is characteristic of PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 11 / 22 PLOS ONE Bee gut bacteria and chalkbrood Fig 6. Chalkbrood bioassays. A chalkbrood fungal plug was placed at the top end of the plate with bacteria streaked at
the bottom and incubated at 18˚C for 10 days. (A) bacterial bee gut isolate 23H1Gw4.8 (no inhibition), (B) bacterial
bee gut isolate 28H4Tw4.20 (inhibition, > 30%), and (C) Pseudomonas strain AN5 (inhibition, > 30%). https://doi.org/10.1371/journal.pone.0238252.g006 Fig 6. Chalkbrood bioassays. A chalkbrood fungal plug was placed at the top end of the plate with bacteria streaked at
the bottom and incubated at 18˚C for 10 days. (A) bacterial bee gut isolate 23H1Gw4.8 (no inhibition), (B) bacterial
bee gut isolate 28H4Tw4.20 (inhibition, > 30%), and (C) Pseudomonas strain AN5 (inhibition, > 30%). https://doi.org/10.1371/journal.pone.0238252.g006 https://doi.org/10.1371/journal.pone.0238252.g006 gluconic acid. Collision-induced dissociation (CID) analysis of this 195-m/z peak revealed glu-
conic acid polymers (S4 Fig), confirming that the peak indeed represents gluconic acid [43]. Using similar methods, we found that the aerobic bee gut isolates DT4 and ET6 also produce
gluconic acid but at a lower level than what was observed for ET2 (i.e., 20 and 10% lower,
respectively). In previous studies, we developed Pseudomonas strain AN5 as an effective biocontrol agent
for the wheat “take-all” pathogen. Gluconic acid production by bacteria and its effect on the chalkbrood
pathogen We further showed that this bacterial strain produces glu-
conic acid and that this metabolite is essential for the suppression of the “take-all” pathogen
[33]. Here, we report that strain AN5 also strongly inhibits the chalkbrood pathogen (% inhibi-
tion, 30%; Fig 6C) in contrast to two independent transposon mutants of AN5, AN5MN1
and AN5MN2 [33] that did not produce gluconic acid and were unable to inhibit the chalk-
brood pathogen (i.e., in our bioassay, the chalkbrood fungus grew over the colonies of these
AN5 mutants). Subsequent experiments, designed to further investigate the effect of gluconic acid on the
chalkbrood pathogen, were conducted with Pseudomonas strain AN5 rather than the aerobic
bee gut isolate ET2, as AN5 produces more gluconic acid than ET2. The switch to strain Pseu-
domonas AN5 allowed us to take advantage of the two AN5 transposon mutants that do not
produce gluconic acid [33]. To investigate how Pseudomonas strain AN5 inhibits chalkbrood,
semi-purified crude bacterial extracts were made from the wild-type strain and the two
mutants, AN5MN1 and AN5MN2. While extracts from the wild-type AN5 strain strongly
inhibited the chalkboard fungus, extracts from the mutant strains showed no inhibition. This
result confirmed our hypothesis that gluconic acid production is essential for the observed
inhibition of the chalkbrood pathogen. Some of the chalkbrood inhibition bioassays were per-
formed on thin PDA plates, so light microscopic studies of the chalkbrood mycelia could be
carried out. After 8 days of incubation at 18˚C, normal fungal growth is characterized by
mycelia that consist of filaments that branch often and have regular septae (Fig 7A). When a
semi-crude extract of the Pseudomonas strain AN5 was added to a paper disc near the fungal
plug, we observed a breakdown of the septae, hyphal lysis, and cytoplasmic leakage (Fig 7B
and 7C), while neither the extracts of the two transposon mutants AN5MN1 and AN5MN2,
nor the addition of water did affect the growth of the chalkbrood mycelia. Genetic characterization of aerobic bee gut bacteria that inhibit the
chalkbrood pathogen Nineteen aerobic bee gut bacterial isolates (Table 1) that strongly inhibited the chalkboard
pathogen, were chosen for identification. We chose these 19 strains to represent different 12 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 PLOS ONE Bee gut bacteria and chalkbrood Fig 7. Bacterial metabolite-mediated cytopathic effects on hyphae of the chalkbrood fungus. Light microscopy
images of chalkbrood mycelia that were grown on thin potato dextrose agar (PDA). (A) Mycelium of an untreated
control; (B, C) mycelia that were treated with a semi-purified crude extract of Pseudomonas strain AN5. In panel B, the
arrows indicate fragmentation due to a breakdown of septae; in panel C, the arrows indicate hyphal lysis and
cytoplasmic leakage. Magnification, 1,000×. Fig 7. Bacterial metabolite-mediated cytopathic effects on hyphae of the chalkbrood fungus. Light microscopy
images of chalkbrood mycelia that were grown on thin potato dextrose agar (PDA). (A) Mycelium of an untreated
control; (B, C) mycelia that were treated with a semi-purified crude extract of Pseudomonas strain AN5. In panel B, the
arrows indicate fragmentation due to a breakdown of septae; in panel C, the arrows indicate hyphal lysis and
cytoplasmic leakage. Magnification, 1,000×. https://doi.org/10.1371/journal.pone.0238252.g007 https://doi.org/10.1371/journal.pone.0238252.g007 geographic locations, Gram staining characteristics, and other properties of interest (e.g., glu-
conic acid production). In each case, a 500-bp region of the 16S rRNA gene was amplified by
PCR with universal primers, Sanger sequenced, and an NCBI Blast search was carried out. The
16S rRNA gene sequence of the aerobic bacterial bee gut isolate 16H10Pw4.9 was found to per-
fectly match that of Bacillus thuringiensis strain bias Z2 (sequence similarity, 100%; E y
g
( q
y,
;
value = 0). Sequences of the other 18 aerobic bee gut bacterial isolates were analyzed in a simi-
lar manner and allowed us to define the following four clusters (sequence similarities > 97%):
cluster I, Bacillus strains; cluster II, enteric strains; cluster III, Macrococcus strains; and cluster
IV, Frigoribacterium and Actinobacterium strains. The phylogenetic relationship of the Gram-
positive Bacillus isolates of cluster I (13 isolates) is shown as a phylogenetic tree (Fig 8). In
some cases, however, analyzing 16S rRNA sequences was not sufficient to determine the phy-
logenetic relationships between very closely related species. Genetic characterization of aerobic bee gut bacteria that inhibit the
chalkbrood pathogen For example, the aerobic bee gut
isolate 16H10Pw4.9 showed 100% sequence identity to Bacillus thuringiensis strain bios Z2,
Bacillus mycoides strain BGSC 6A13, and Bacillus cereus strain JSCtot9-2; further sequence
analyses using other gene sequences (e.g., gyrase sequences) is required to identify the closest
relative of this isolate. Cluster II contains four isolates with sequence similarities to Gram-neg-
ative enteric bacteria. Two isolates, 16H13Pw2.4 and 16H14Ew3.3, showed a high degree of
sequence similarity to Hafnia alvei (> 97%; E value = 0). Isolate 14H12Pw3.4 showed sequence
similarity to the Enterobacter species Leclercia adecarboxylata (99%; E value = 0), and the
sequence of isolate 20H2Ew2.1 was identical to an Enterobacter aerogenes sequence (100%; E
value = 0). The phylogenetic tree of cluster II is shown in Fig 9. Similar phylogenetic analyses
were carried out with clusters III and IV. The only cluster III isolate, ET2, was identical to
Macrococcus hajekii (100%; E value = 0), and the only cluster IV isolate, DT4, was identical to
Frigoribacterium species (100%; E value = 0). Bootstrap values of > 50% for all branches (of all
trees) indicate the robustness of the analyses and gives strong validity to the phylogenetic rela-
tionships observed. Discussion The European honey bee gut has a well-defined “core” microbiome that contains bacteria
from only a few (i.e., less than 10) phylotypes [44, 45]. According to metagenomic studies Gil-
liamella, Snodgrassella, and Lactobacillus ssp. were the most frequently found species in worker
bees; Bifidobacterium, Frischella, Bartonella, Parasaccharibacter, and Gluconobacter were less
frequently found, but these genera are still regarded as part of the “core” microbiome [17, 21–
25, 44, 45]. These mostly anaerobic, facultative anaerobic or microaerophilic bacteria have co- PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 13 / 22 PLOS ONE Bee gut bacteria and chalkbrood evolved with the honey bee host [44], and their presence is considered to be important for
honey bee health [46]. The bacteria in the honey bee gut play important roles in digestion (e.g.,
carbohydrate utilization), nutrition, weight gain, preservation of pollen, disease resistance,
Fig 8. Phylogenetic analysis of 16S rRNA sequences (cluster I; Bacillus strains). Complementary DNA was
amplified from aerobic bacteria isolated from the gut of nurse bees (Apis millifera) from Australia, sequenced, and
sequences were aligned using the Fast Fourier Transform (MAFFT). A maximum likelihood phylogenetic tree was
generated using PhyML3.0 and the general time reversible (GTR) evolutionary model. Bootstrap values detected for
100 replicates are shown near the nodes. The scale bar represents the change in nucleotides of the sequence (i.e.,
genetic variation for the length of scale). https://doi.org/10.1371/journal.pone.0238252.g008
PLOS ONE
Bee gut bacteria and chalkbrood Fig 8. Phylogenetic analysis of 16S rRNA sequences (cluster I; Bacillus strains). Complementary DNA was
amplified from aerobic bacteria isolated from the gut of nurse bees (Apis millifera) from Australia, sequenced, and
sequences were aligned using the Fast Fourier Transform (MAFFT). A maximum likelihood phylogenetic tree was
generated using PhyML3.0 and the general time reversible (GTR) evolutionary model. Bootstrap values detected for
100 replicates are shown near the nodes. The scale bar represents the change in nucleotides of the sequence (i.e.,
genetic variation for the length of scale). https://doi org/10 1371/journal pone 0238252 g008 Fig 8. Phylogenetic analysis of 16S rRNA sequences (cluster I; Bacillus strains). Complementary DNA was
amplified from aerobic bacteria isolated from the gut of nurse bees (Apis millifera) from Australia, sequenced, and
sequences were aligned using the Fast Fourier Transform (MAFFT). A maximum likelihood phylogenetic tree was
generated using PhyML3.0 and the general time reversible (GTR) evolutionary model. Bootstrap values detected for
100 replicates are shown near the nodes. Discussion The scale bar represents the change in nucleotides of the sequence (i.e.,
genetic variation for the length of scale). Fig 8. Phylogenetic analysis of 16S rRNA sequences (cluster I; Bacillus strains). Complementary DNA was
amplified from aerobic bacteria isolated from the gut of nurse bees (Apis millifera) from Australia, sequenced, and
sequences were aligned using the Fast Fourier Transform (MAFFT). A maximum likelihood phylogenetic tree was
generated using PhyML3.0 and the general time reversible (GTR) evolutionary model. Bootstrap values detected for
100 replicates are shown near the nodes. The scale bar represents the change in nucleotides of the sequence (i.e.,
genetic variation for the length of scale). https://doi.org/10.1371/journal.pone.0238252.g008 evolved with the honey bee host [44], and their presence is considered to be important for
honey bee health [46]. The bacteria in the honey bee gut play important roles in digestion (e.g.,
carbohydrate utilization), nutrition, weight gain, preservation of pollen, disease resistance, 14 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 pheromone production, reproduction, endocrine signaling, immune function, and honey pro-
duction [17–19, 47]. Furthermore, gut bacteria have the ability to inhibit pathogens, such as
the chalkbrood fungus [20]. Like bacteria in the human intestine, most bee bacteria are found
in the distal part of the gut where most occupy specific niches [17]. Bees exchange gut bacteria
Fig 9. Phylogenetic analysis of 16S rRNA sequences (cluster II; enteric bacteria). Complementary DNA was
amplified from aerobic bacteria isolated from the gut of nurse bees (Apis millifera) from Australia, sequenced, and
sequences were aligned using the Fast Fourier Transform (MAFFT). A maximum likelihood phylogenetic tree was
generated using PhyML3.0 and the general time reversible (GTR) evolutionary model. Bootstrap values detected for
100 replicates are shown near the nodes. The scale bar represents the change in nucleotides of the sequence (i.e.,
genetic variation for the length of scale). https://doi.org/10.1371/journal.pone.0238252.g009
S ONE
Bee gut bacteria and chalkbrood PLOS ONE Bee gut bacteria and chalkbrood Fig 9. Phylogenetic analysis of 16S rRNA sequences (cluster II; enteric bacteria). Complementary DNA was
amplified from aerobic bacteria isolated from the gut of nurse bees (Apis millifera) from Australia, sequenced, and
sequences were aligned using the Fast Fourier Transform (MAFFT). A maximum likelihood phylogenetic tree was
generated using PhyML3.0 and the general time reversible (GTR) evolutionary model. Bootstrap values detected for
100 replicates are shown near the nodes. Discussion The gut microbiota in bees from Australia has not been extensively tested, with a notable
exception of a recent study that investigated the microbiome composition in European honey
bees and stingless bees (tribe Meliponini) from Queensland [45]. We isolated and character-
ized aerobic gut bacteria from European honey bees (nurse bees) that were collected from
healthy hives, chalkbrood-infested hives and from hives that had recently recovered from
chalkbrood. We also surveyed honey bees that were especially bred for enhanced hygienic
behavior [41]. In the Australian late spring, summer and early autumn, bees from healthy colo-
nies had 107–108 aerobic bacteria per g of bee gut (counted using TSA, a general purpose, non-
selective medium for the cultivation and isolation of fastidious and non-fastidious bacteria;
TSA has been noted as useful for this purpose [50]). Colonies with signs of the chalkbrood dis-
ease (from the same apiary) had significantly fewer aerobic bee gut bacteria (104–105 bacteria
per g) than bees from healthy colonies. We also followed bacterial numbers in chalkbrood-dis-
eased colonies over time, which revealed that bacterial numbers recovered when a colony no
longer showed signs of chalkbrood. This observation establishes a direct correlation between
the number of culturable aerobic bacteria in the gut of individual nurse bees and the health sta-
tus of a colony. The decrease in aerobic gut bacterial numbers of bees from chalkbrood- Nurse bees combat disease by removing infected larvae from the hive [54], a behavior that
also promotes recovery from chalkbrood [57]. Bees from the West Australian “Better Bees”
program are descendants from Apis mellifera ligustica breeding stocks [58] that were selec-
tively bred for high honey production, good brood viability, lack of aggressiveness, low inci-
dence of brood diseases, and high colony population size [41]. The “Better Bees” program has
been somewhat successful in providing West Australian beekeepers with quality queens for
improved honey production [59]. However, a subsequent report states that the hygienic behav-
ior of such bee lines can vary and may not be a reliable character [60]. We found that bees
from the “Better Bees” program had consistently significantly higher numbers of aerobic bee
gut bacteria compared with “normal” bees from apiaries in the same area. It is tempting to
speculate that “Better Bees” possess an enhanced metabolic rate because they have higher con-
centrations of carbon sources in their gut, which allows for a greater number of gut bacteria. Discussion The scale bar represents the change in nucleotides of the sequence (i.e.,
genetic variation for the length of scale). https://doi.org/10.1371/journal.pone.0238252.g009 Fig 9. Phylogenetic analysis of 16S rRNA sequences (cluster II; enteric bacteria). Complementary DNA was
amplified from aerobic bacteria isolated from the gut of nurse bees (Apis millifera) from Australia, sequenced, and
sequences were aligned using the Fast Fourier Transform (MAFFT). A maximum likelihood phylogenetic tree was
generated using PhyML3.0 and the general time reversible (GTR) evolutionary model. Bootstrap values detected for
100 replicates are shown near the nodes. The scale bar represents the change in nucleotides of the sequence (i.e.,
genetic variation for the length of scale). https://doi.org/10.1371/journal.pone.0238252.g009 Fig 9. Phylogenetic analysis of 16S rRNA sequences (cluster II; enteric bacteria). Complementary DNA was
amplified from aerobic bacteria isolated from the gut of nurse bees (Apis millifera) from Australia, sequenced, and
sequences were aligned using the Fast Fourier Transform (MAFFT). A maximum likelihood phylogenetic tree was
generated using PhyML3.0 and the general time reversible (GTR) evolutionary model. Bootstrap values detected for
100 replicates are shown near the nodes. The scale bar represents the change in nucleotides of the sequence (i.e.,
genetic variation for the length of scale). https://doi.org/10.1371/journal.pone.0238252.g009 https://doi.org/10.1371/journal.pone.0238252.g009 pheromone production, reproduction, endocrine signaling, immune function, and honey pro-
duction [17–19, 47]. Furthermore, gut bacteria have the ability to inhibit pathogens, such as
the chalkbrood fungus [20]. Like bacteria in the human intestine, most bee bacteria are found
in the distal part of the gut where most occupy specific niches [17]. Bees exchange gut bacteria pheromone production, reproduction, endocrine signaling, immune function, and honey pro-
duction [17–19, 47]. Furthermore, gut bacteria have the ability to inhibit pathogens, such as
the chalkbrood fungus [20]. Like bacteria in the human intestine, most bee bacteria are found
in the distal part of the gut where most occupy specific niches [17]. Bees exchange gut bacteria PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 15 / 22 PLOS ONE Bee gut bacteria and chalkbrood with other bees of the same colony through social interactions [28, 48]. It is likely that, as with
humans, changes to the bee gut microbiota can lead to a reduced host fitness [49]. g
g
The gut microbiota in bees from Australia has not been extensively tested, with a notable
exception of a recent study that investigated the microbiome composition in European honey
bees and stingless bees (tribe Meliponini) from Queensland [45]. Discussion We isolated and character-
ized aerobic gut bacteria from European honey bees (nurse bees) that were collected from
healthy hives, chalkbrood-infested hives and from hives that had recently recovered from
chalkbrood. We also surveyed honey bees that were especially bred for enhanced hygienic
behavior [41]. In the Australian late spring, summer and early autumn, bees from healthy colo-
nies had 107–108 aerobic bacteria per g of bee gut (counted using TSA, a general purpose, non-
selective medium for the cultivation and isolation of fastidious and non-fastidious bacteria;
TSA has been noted as useful for this purpose [50]). Colonies with signs of the chalkbrood dis-
ease (from the same apiary) had significantly fewer aerobic bee gut bacteria (104–105 bacteria
per g) than bees from healthy colonies. We also followed bacterial numbers in chalkbrood-dis-
eased colonies over time, which revealed that bacterial numbers recovered when a colony no
longer showed signs of chalkbrood. This observation establishes a direct correlation between
the number of culturable aerobic bacteria in the gut of individual nurse bees and the health sta-
tus of a colony. The decrease in aerobic gut bacterial numbers of bees from chalkbrood-
infected colonies could be due to homeostatic imbalances caused by the presence of the chalk-
brood fungus, i.e., metabolic changes due to chalkbrood infection may create conditions that
no longer support large bacterial numbers and a diverse microbial flora. A critical factor could
be the depletion of nutrients caused by the nutritional demands of growing chalkbrood myce-
lia [51]. Furthermore, the build-up of chalkbrood mycelia in the gut of infected nurse bees
restricts the amount of space that is available for bacteria [52]. Our findings suggest that the
number of aerobic gut bacteria can be used as a diagnostic tool to determine the relative health
status of a colony, independent and potentially ahead of “classic” chalkbrood disease signs
(such as cells with dead larvae, or mummified larvae in front of the hive; [53, 54]). Future stud-
ies will determine whether other fungal bee pathogens have a similar effect on aerobic gut bac-
terial numbers. An interesting candidate is Nosema apis, a microsporidian fungus that, until
recently, was classified as a protozoan parasite [55, 56]. PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 Discussion In this study, we did not look at the role of hygienic behavior in disease resistance. It would be
interesting to determine aerobic bee gut bacterial numbers in other bee lines which have genet-
ically distinct queens [61]. Our preliminary genetic characterization of 19 Australian chalkbrood-inhibiting isolates
from different Australian locations identified four species clusters, i.e., Bacillus ssp. (cluster I),
enteric strains (cluster II), Macrococcus sp. (cluster III), and Frigoribacterium sp. (cluster IV). Bacillus ssp. were the most frequently isolated bacteria in this study; we found Bacillus cereus, PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 16 / 22 PLOS ONE Bee gut bacteria and chalkbrood Bacillus senegalensis, Bacillus pumilus, Bacillus flexus, Bacillus megaterium, Bacillus thuringien-
sis, and Bacillus macroides in the honey bee gut. Bacillus ssp. and enteric bacteria (Enterobacter
aerogenes) have previously been isolated from worker bees in the United States [50, 62, 63]. The isolation of Bacillus cereus should be of particular interest for the bee industry, because a
strain of this species that was previously isolated from honey bees inhibits Paenibacillus larvae,
the etiologic agent of European foul brood [64]. The isolation of Enterobacter aerogenes in
Australian honey bees confirms previous reports of E. aerogenes in the bee gut flora of foraging
bees from Tucson, Arizona [62, 63]. Interestingly, our cluster III and IV isolates (Macrococcus
hajekii and Frigoribacterium sp.) have not previously been found in bees. Whether these bacte-
ria represent a uniquely Australian contribution to the microbiome of honey bees remains to
be seen. This study did not aim at isolating the mostly anaerobic or microaerophilic “core” micro-
biome bacteria. Instead, we focused on facultative anaerobes and aerobes, the so-called “envi-
ronmental” gut bacteria. We show that these bacteria occur in high numbers (i.e., 107–108
CFUs per gram of bee gut) during the foraging season for honey bees. This observation is
based on the analysis of more than 400 bees from nearly 100 colonies that were sampled all
around Australia, representing different climate zones and pollen/nectar sources. The majority
of these “environmental” bacteria is brought into the hive by foraging worker bees who pass
them on to nurse bees trough food and social interactions [65]. Gluconic acid is a simple sugar with anti-fungal properties that can be utilized for the bio-
control of the take-all wheat pathogen [33]. Here, we report that the gluconic acid producing
Pseudomonas strain AN5 inhibits the chalkbrood fungus. Discussion By using transposon independent
mutants that do not produce gluconic acid, we show that the production of gluconic acid is
essential for the antifungal activity of strain AN5. We further demonstrate that Pseudomonas
strain AN5 extracts act on the chalkbrood fungus through hyphal lysis and cytoplasmic leak-
age. Our observation that Maccrococcus hajekii, Frigoribacterium sp., and Bacillus senegalensis
isolates also produced gluconic acid suggests a similar modus operandi. However, these strains
produced less gluconic acid than Pseudomonas strain AN5 but exhibited a similar degree of
anti-fungal activity. Therefore, gluconic acid may not be the only metabolite by which bee gut
isolates inhibit the chalkbrood pathogen. Interestingly, gluconic acid is considered to be the
honey component that is largely responsible for the anti-microbial properties of honey [66]. Bees produce gluconic acid in the hypopharyngeal glands and in the gut with the help of gut
bacteria [67]; however, more research is needed to understand how much gut bacteria contrib-
ute to the total gluconic acid production. Furthermore, it will be interesting to determine if
and to what extent the bacterial gluconic acid production contributes to the resistance against
bee pathogens. Supporting information S1 Fig. Flowchart for the isolation and characterization of aerobic gut bacteria in Euro-
pean honey bees (Apis millifera) from Australia. (TIF) S1 Fig. Flowchart for the isolation and characterization of aerobic gut bacteria in Euro-
pean honey bees (Apis millifera) from Australia. (TIF) S2 Fig. “Environmental” bacteria from the gut of a healthy European honey nurse bee
(Apis millifera) from Australia. The gut of the bee (without the crop) was homogenized in 1
ml of nutrient broth, diluted, and aliquots of the homogenate was plated on (A) tryptic soy
agar (TSA), (B) eosin methylene blue agar (EMB), (C) glucose calcium carbonate media
(G-CaCO3), and (D) modified Gould S1 media (mS1 Gould). The photos show microbial colo-
nies after 2 days of incubation at 25˚C under aerobic conditions. (TIF) S2 Fig. “Environmental” bacteria from the gut of a healthy European honey nurse bee
(Apis millifera) from Australia. The gut of the bee (without the crop) was homogenized in 1
ml of nutrient broth, diluted, and aliquots of the homogenate was plated on (A) tryptic soy
agar (TSA), (B) eosin methylene blue agar (EMB), (C) glucose calcium carbonate media
(G-CaCO3), and (D) modified Gould S1 media (mS1 Gould). The photos show microbial colo-
nies after 2 days of incubation at 25˚C under aerobic conditions. (TIF) S3 Fig. Detection of acid production by bromocresol purple. “Environmental” bee gut iso-
lates 15H12Ew1.1 (A) and 21H2Ew2.7 (B) were grown on potato dextrose agar (PDA) supple-
mented with bromocresol purple (15 mg/L). Plates were incubated at 25˚C for 2 days under
aerobic conditions. Acid production is indicated by a color change from purple (pH > 6.8) to
yellow (pH < 5.2). (TIF) S3 Fig. Detection of acid production by bromocresol purple. “Environmental” bee gut iso-
lates 15H12Ew1.1 (A) and 21H2Ew2.7 (B) were grown on potato dextrose agar (PDA) supple-
mented with bromocresol purple (15 mg/L). Plates were incubated at 25˚C for 2 days under
aerobic conditions. Acid production is indicated by a color change from purple (pH > 6.8) to
yellow (pH < 5.2). (TIF) S4 Fig. Detection of gluconic acid by mass spectrum analysis. (A) A hydrophilic extract of
the nurse bee gut isolate ET2 was analyzed using a VG QUATTRO II mass spectrometer (with
the arrow indicating the 195 m/z peak characteristic of gluconic acid). Supporting information (B) Results of collision-
induced dissociation (CID) analysis of the 195 m/z peak in panel B, confirms the presence of
gluconic acid polymers. (TIF) Conclusion In this study, we used culturable aerobic and facultative anaerobic honey bee bacteria as a sim-
ple tool to monitor colony health. The method allows the rapid analysis of many samples and
results are available within two days. Our findings suggest that decreasing numbers of cultur-
able aerobic gut bacteria in nurse bees are a prognostic marker for the outbreak of chalkbrood
and that increasing numbers herald recovery. Therefore, counting bacteria could be developed
as a rapid test for apiarists that allows to detect deteriorating colony health before other disease
signs become apparent. Furthermore, our results suggest that chalkbrood-inhibiting bacteria
from the bee gut could be used to develop a probiotic treatment for chalkbrood and potentially
other fungal bee diseases. PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
August 28, 2020 17 / 22 PLOS ONE Bee gut bacteria and chalkbrood Acknowledgments We thank Margaret Hilton and Phil Jackson (Australian National University) for carrying out
the mass spectrometry analysis. We thank Mark Gibbs for help with bioinformatics and Rob
Manning for sampling honey bees from the “Better Bee” program. Kerry Mills (University of
Canberra) is thanked for feedback on the manuscript. We are indebted to apiarists around
Australia who provided many of the honey bees that were analyzed in this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0238252
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ing Apis mellifera mellifera L. bees. Microbiol. 2008; 77(3): 373–9. https://doi.org/10.1134/
S0026261708030181 64. Evans JD, Armstrong T-N. Antagonistic interactions between honey bee bacterial symbionts and impli-
cations for disease. BMC Ecol. 2006; 6(1): 4. https://doi.org/10.1186/1472-6785-6-4 65. Corby-Harris V, Maes P, Anderson KE. The bacterial communities associated with honey bee (Apis
mellifera) foragers. PLOS ONE. 2014; 9(4): e95056. https://doi.org/10.1371/journal.pone.0095056
PMID: 24740297 66. Kwakman PHS, Zaat SAJ. Antibacterial components of honey. IUBMB Life. 2012; 64(1): 48–55. https://
doi.org/10.1002/iub.578 PMID: 22095907 66. Kwakman PHS, Zaat SAJ. Antibacterial components of honey. IUBMB Life. 2012; 64(1): 48–55. https://
doi.org/10.1002/iub.578 PMID: 22095907 67. Wright GA, Nicolson SW, Shafir S. Nutritional physiology and ecology of honey bees. Annu Rev Ento-
mol. 2018; 63. https://doi.org/10.1146/annurev-ento-020117-043423 67. Wright GA, Nicolson SW, Shafir S. Nutritional physiology and ecology of honey bees. Annu Rev Ento-
mol. 2018; 63. https://doi.org/10.1146/annurev-ento-020117-043423 22 / 22
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https://openalex.org/W4247268198
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https://zenodo.org/record/1825773/files/article.pdf
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English
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Introduction to the Study of the Fundamental Cause of Splanchn Optosis — Abdominal Incompetence: A Developmental Factor
|
The Boston medical and surgical journal/Boston medical and surgical journal
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References. 1 Brit. Med. Jour., Oct. 1903, p. 1077. 2 Lancet, Aug. 27, 1904. 3 Congrès des Alien & Neurol., Bordeaux, Août, 1895. « Deut. Med. Woch., 1899, p. 627. « Neurol. Centrabl., 1901, No. 22. 6 Munch. Med. Woch., 1902, No. 20. 7 Ibid., 1903. No. 4. 8 Lyon: Med., 1903, Nov. 29. 9 Munch. Med. Woch., 1903, No. 9. 10 Deutsch. Med. Woch., 1903, No. 20. »Rev. Neurol., 1904, p. 1109. "Med. Klinic, 1904, Dec. 13 Die. Med. Woch., 1904, No. 37. 14 Jour. Am. Med. Assoc, 1905, April 22. 15 Medicine, Detroit, 1905. 16 Presse Médicale, Paris, Nov., 1905. "Munch, Med. Woch., May 2, 1905. 18 Jour. Am. Med. Assoc., Feb. 17, 1906. this body wall by muscles
and
integuments. This connection of the extremities with the body
wall decreases as one rises in the animal series
till in their highest development these extremi-
ties become specialized to nearly complete inde-
pendence of the trunk; the latter serving only as
a fixed point upon which all the more highly
specialized
movements of the extremities
are
based. This fixed point is utilized through the
large muscles which pass from the trunk to the
extremity. And these connecting muscles
re-
quire a certain amount of trunk stability to fur-
nish a point of departure for their activities. I
i t
ed Jour., Oct. 2 Lancet, Aug. 27, 1904. 3
d Rev. Neurol., 1904, p. 11
"Med. Klinic, 1904, Dec. Med. Klinic, 1904, Dec. 13 Die. Med. Woch., 1904, No. 37. A e
ed Woch., 1904, No. 37. 14 Jour. Am. Med. Assoc
1905, April 22. M di i Jour. Med. Assoc, 1905, April 22. 15 Medicine, Detroit
1905. ed Assoc,
15 Medicine, Detroit, 1905. Médi
l ed c e, Detroit, 1905. 16 Presse Médicale, Paris, Nov., 1905. M d Médicale, Paris, Nov.,
"Munch, Med. Woch., May 2, 1905. Munch,
Woch., May 2, 1905. 18 Jour. Am. Med. Assoc., Feb. 17, 1906. p
departure
In proportion as the evolution of the extremity
is imperfect or incomplete, clinical observation
shows that stress upon it is carried backward
to its attachment to the trunk; and if this attach-
ment is deficient in strength
or coordination,
the stress is carried still further backward, and
the body wall itself strives to make up for the
insufficiency of the extremity to the demand
made upon it. OUTLINE
OF
EVOLUTION
OF
SPECIFIC
HUMAN
ABDOMEN. Correlated with evolution of pelvis, thorax and
extremities;
especially with extension, adduction
and forward rotation of the lower extremities,
so
that the patella and toes point forward. y
y
conveyed
Examples of this will be given later. It is
mentioned here because it is a manifestation of
a law which seems to explain the action which
insufficiently developed (in strength
or
coor-
dination) extremities, both upper and lower, but
especially the lower, have in causing visceral
ptosis, this being one of the most common causes
of this affection. patella
point
The final distinguishing characteristic of the
human genus is the full extension of the lower
extremities in the plane of the vertical trunk,
the dorsal and ventral surfaces of which tend
constantly to approach each other in approxi-
mately parallel planes. The
converse of this is also observed. The
circle of insufficiency and deformity may begin
with abdominal incompetence or visceral ptosis,
causing inefficiency of the trunk as a point of
departure for developing activities of the
ex-
tremities, leading to inefficiency
or imperfect
development of these extremities. mately parallel planes. This extension of the lower extremities upon
the vertical trunk (a) Removes the support of the erect (sitting)
pelvis from the abdominal viscera. (b) R
f p
(b) Removes the support of the flexed thighs
which supplement the erect pelvis in supporting
the abdomen. p
Those portions of the body wall which make
up the abdomen are easily and early involved
in attempts to compensate for weakness of the
extremities. (c) Adds forward rotation of the pelvis and
anterior convexity of the lumbar spine;
both
of these conditions throwing the viscera forward
and so facilitating their descent, directly through
gravity and indirectly by increasing the stress on
the supporting anterior and posterior abdominal
walls, and so impairing the supporting power of
these walls. If the abdominal walls have developed com-
petence, they aid the body wall in meeting these
demands, and the stress is met. ,
If the abdominal walls have not developed
competence, they share in the stress, and are, by
so much, diverted from their function of sur>
porting and retaining the viscera. In this way,
varying degrees of visceral ptosis
are due to
weakness of the extremities or of other portions
of the body wall. The converse of this is also
observed, as already noted. OUTLINE
OF
EVOLUTION
OF
SPECIFIC
HUMAN
ABDOMEN. (d) Alters the conditions of respiratory equilib-
rium, because whatever affects the supporting
power of the walls of the abdomen affects the
action of the diaphragm, which latter is only
another abdominal wall
— the superior wall or
roof. If
these
various changes
are
not
counter-
balanced
by compensatory
changes
in
the
anterior and posterior abdominal walls, they
lead to further visceral displacement. The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by
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For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine Downloaded from nejm org at SAN DIEGO (UCSD) on June In this manner may be produced
abdominal incompetence,
visceral
ptosis
and
deformities of the spine, pelvis, thorax, and even
of the other extremities, since insufficiency of
the body wall may be further conveyed to these. EFFECT
OF
ABDOMINAL
INCOMPETENCE
ON
THE
MECHANICS OF RESPIRATION. increasingly
These changes in the thorax anteriorly lead to
two main types
of
changes
posteriorly:
(a)
The posterior wall of the thorax attempts to
parallel the anterior wall;
the ribs retract pos-
teriorly, and an increased lumbo-thoracic anterior
convexity appears. In the supine position, this
forward and upward rotation of the whole thorax
may be seen with each inspiration. This type of
respiration is favored by the conformation of
the trunk, especially when the lower extremities
are extended, since the anatomical paths leading
to it may be developed at term,
(b) The ten-
dency towards (a) is antagonized by
a slight
lumbar anterior convexity and a slight forward
rotation of the pelvis;
and in such patients
the attempts of the posterior wall of the thorax
to parallel the anterior, leads to hyperextension
of the thoracic spine, or lumbo-thoracic junction,
or upper lumbar spine. anatomical
and
physiological
variations
predisposing some of the viscera to
dis-
placement;
and
the
relation
of
this
predisposition to certain frequent patho-
logical conditions. Experiments and dissections show that ab-
dominal
incompetence
opens
the
anatomical
paths for the production of displacements of
the
abdominal viscera, leading
to
conditions
making for repeated traumatisms, through the
action of gravity, and of traction of the viscera
on each other and on the connecting peritoneal
folds which contain the nerves and blood and
lymph vessels. Th
h y p
These observations support the clinical hy-
pothesis that
abdominal incompetence
is the
primary factor in the development of the common
surgical diseases of the upper abdomen
— the
stomach, duodenum, gall bladder and bile ducts,
jejuno-ileum, pancreas and kidneys (especially
the right), being the
most important viscera
affected. pp
sp e
This change appears to be the physiological
one, developing spontaneously in the healthy
child beginning to stand and walk alone. (2) Thi beg
g
(2) This may be called the posterior type. EFFECT
OF
ABDOMINAL
INCOMPETENCE
ON
THE
MECHANICS OF RESPIRATION. Clinical observation shows two main types of
respiration :
h
i displacement. Soon after the enclosure of the viscera by
the body walls,
the four extremities appear. Whether viewed as outgrowths of, or additions
to, the body wall, these extremities are, through-
out the vertebrate series, more or less bound to respiration
(1) This may be called the anterior type. The
abdomen is incompetent;
the diaphragm and
inspiring lungs
make marked
downward and
forward excursion into the abdomen, displacing respiration
(1) This may be called the anterior type. The
abdomen is incompetent;
the diaphragm and
inspiring lungs
make marked
downward and
forward excursion into the abdomen, displacing anterior type; but the advanced stage of develop-
ment of the back muscles, and the supine position
strongly favor the efforts towards the posterior
type which steadily gains. the viscera downward and forward, the viscera
are also displaced upward
and forward;
the
sternum rotates
on its superior extremity, up-
ward and forward, carrying with it its attached
ribs; the bony thorax grows shorter and broader
in the anterior plane, its anterior wall moving
upward
and forward towards the horizontal;
backward expansion of the trunk is diminished
or may be absent;
the ribs move forward and
upward, they
move outward very slightly, ex-
cept in their anterior third or half; expiration
becomes increasingly inefficient. the viscera downward and forward, the viscera
are also displaced upward
and forward;
the
sternum rotates
on its superior extremity, up-
ward and forward, carrying with it its attached
ribs; the bony thorax grows shorter and broader
in the anterior plane, its anterior wall moving
upward
and forward towards the horizontal;
backward expansion of the trunk is diminished
or may be absent;
the ribs move forward and
upward, they
move outward very slightly, ex-
cept in their anterior third or half; expiration
becomes increasingly inefficient. yp
y ga s
As soon as the sitting position is assumed, the
pull of the viscera and the stress on the back,
which is struggling to support the trunk in the
vertical plane, again favor the anterior type. i p a e, aga
type
When the sitting is changed to the standing
position the stress is still greater, and the anterior
type becomes increasingly easier. Nevertheless
the struggle towards the evolution of the pos-
terior type is always present. The result
is the
development of many variations of combinations
of both types. EFFECT
OF
ABDOMINAL
INCOMPETENCE
ON
THE
MECHANICS OF RESPIRATION. The
abdomen is competent; the whole posterior wall
of the trunk
moves backward in inspiration,
backward and upward, and backward and down-
ward; the lateral walls move backward and out-
ward, as well as upward and downward; there
is relatively slight excursion of the diaphragm
and inspiring lungs downward and forward into
the abdomen, and this
occurs mainly in the
upper zone or epigastrium; the sternum and ribs
tend to rotate upward and forward, but this
tendency is antagonized by the downward and
outward pull of the abdominal muscles, and by
the backward as well as the outward, and upward
and downward pull of the posterior and postero-
lateral walls;
this tends to make the anterior
wall approach a plane more or less parallel with
the posterior. Th
d
i SPONTANEOUS
TENDENCY
OF
THE
TRUNK
AND
EXTREMITIES TOWARDS THE DEVELOPMENT OF
SUCH STRUCTURAL VARIATIONS AS ARE CALLED
PATHOLOGICAL,"
DEVELOPMENTAL DEFORMITIES. g
p
y
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by SPONTANEOUS
TENDENCY
OF
THE
TRUNK
AND
EXTREMITIES TOWARDS THE DEVELOPMENT OF
SUCH STRUCTURAL VARIATIONS AS ARE CALLED
PATHOLOGICAL,"
DEVELOPMENTAL DEFORMITIES. ing
by
(1) Introduction to the study of the prenata
evolution of the abdomen, with special referent
to the status at term. Report of Research Fel
lowship of the Society of Lying-in Hospital o
New York, 1903-04 (in press). All grades of variations of these two types of
figures are observed, including exaggerations of
the tendencies of both;
including also transi-
tional figures showing passage from one type to
the other. York,
(
press). (2) Introduction to the study of the postnata
evolution of the abdomen (in preparation). Cli i
l
bd
i
l o ,
(
p ess)
(2) Introduction to the study of the postnata
evolution of the abdomen (in preparation). These variations all resemble each other in that
the equilibrium attained is very unstable, and,
therefore, these variations are constantly inter-
changing details to a greater or less extent. (
preparation). (3) Clinical study of abdominal incompetent
and visceral ptosis (in preparation). (
p epa at o )
(3) Clinical study of abdominal incompetent
and visceral ptosis (in preparation). g g
g eate
The first mentioned type appears to be basic
or primary because it is always present at birth
and in early life; because it is the type towards
which the greatest number of figures tend;
be-
cause it is found most often in strong and robust
individuals; and because the equilibrium of this
type is, as a whole, the most stable. EVOLUTION TOWARDS TWO MAIN TYPES OF HUMAI*
FIGURES,
DEPENDING
UPON
VARIATIONS
IÏ
THESE CORRELATIONS. Type
1. Broad
lower thorax;
slight
lumbo-
sacral anterior convexity; slight forward rotation
of pelvis. h type s,
whole,
The second type appears to be the variant,
secondary, aberrant type because it is
never
present at birth or in early life;
because it is
produced from the basic type at a subsequent
age; because it is most often found in individuals
with a weak musculature
or presenting other
evidences of weakened or poor nutrition. Fur-
thermore, it tends spontaneously to approach
the basic type whenever nutrition is improved,
general muscular activity increased, and when
certain arbitrary conditions are removed, leaving
the figure to the spontaneous modeling of all the
muscles. On the other hand, the equilibrium of
this type is very unstable;
it tends constantly
to vary under slight variations in the correlative
factors mentioned, and to pass readily into such
pathological variations
as result in what
are
called deformities. of pelvis. Type
2. Retracted
lower
thorax;
markea
lumbo-sacral anterior convexity;
marked forward
rotation of pelvis. of pelvis. Type
2. Retracted
lower
thorax;
markea
lumbo-sacral anterior convexity;
marked forward
rotation of pelvis. of pelvis. This type must be considered pathological
— a
variety of developmental deformity. It presents
a
retraction of the trunk walls, with a proportionate
degree of visceral ptosis
en
masse
— a condition
which predisposes to various forms of individual
visceral ptosis ptosis. Correlations towards which each type tends: In
abdomen,
thorax,
lower
extremities,
respiratory
mechanics and specialized activities. spec a ed
According as an equilibrium among the factors
enumerated is reached by one or other varying
paths of compensation, study of the ascent of
the trunk to the vertical plane, and the extension
of the lower extremities in the plane of this
trunk, shows a spontaneous development of two
main types of the human figure. Clinical observation shows that competence
of the abdominal
walls
increases throughout
childhood, and in selected cases is complete before
the age of puberty. It also shows the relation
of competence of the abdomen to the develop-
ment of the rest of the body walls and the extremi-
ties. It
is
well-established
that
during
this
period of development deformities of the spine,
thorax, pelvis and extremities appear, and the
evidence at present accumulated seems to show
that these deformities develop along the lines just
described. g
p
y
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. SPONTANEOUS
TENDENCY
OF
THE
TRUNK
AND
EXTREMITIES TOWARDS THE DEVELOPMENT OF
SUCH STRUCTURAL VARIATIONS AS ARE CALLED
PATHOLOGICAL,"
DEVELOPMENTAL DEFORMITIES. Dissections show that at birth a lumbo-sacral
anterior convexity is present; that an anatomi-
cal condition of spontaneous flexion, adduction
and outward rotation of the lower extremities
(thighs) is present;
that the lower extremities
and the pelvis are united at such an angle that
in proportion
as the lower extremities (thighs)
are flexed, etc., the lumbo-sacral anterior con-
vexity is reduced to a minimum or successivelv
obliterated and converted into an anterior con-
cavity; that in proportion as the lower extremi-
ties (thighs) are extended, adducted and rotated
forward, the lumbo-sacral anterior convexity is
increased
and
extended through
(1)
forward
rotation of the united pelvic bones and sacrum,
and
(2) through the attachment of the fifth
lumbar vertebra to the sacrum
and ilia,
the
supraincumbent lumbar (or even the thoracic)
vertebrae are carried into anterior convexity, pro-
portionate to their mobility. h
l
i
f h p
The predominant forward and upward and
forward and downward movement of the anterior
type is now translated into a backward and up-
ward, and backward and downward movement. The outward
movement of the antero-lateral
planes has become translated into an outward
movement of the postero-lateral planes. I
th
i p
p
In the anterior type the anterior wall models
the trunk;
in the posterior type, the posterior
wall models it. A
bi h
i
i At birth, respiration is modified by all the
factors enumerated and by many others. It is
therefore struggling and incoördinated. O
th
h l
i p
y
For the relation of the postnatal modification
of these conditions to the evolution of the trunk
and the developmental deformities enumerated,
as well
as the details of the above-mentioned gg
g
On the whole, it most nearly approaches the visceral experiments,
dissections
and
clinica
observations, the reader is referred to the follow
ing articles by the writer. visceral experiments,
dissections
and
clinica
observations, the reader is referred to the follow
ing articles by the writer. inclination of the pelvis, and usually a more or
less well-developed incompetence of the abdominal
walls. ing
by
(1) Introduction to the study of the prenata
evolution of the abdomen, with special referent
to the status at term. Report of Research Fel
lowship of the Society of Lying-in Hospital o
New York, 1903-04 (in press). The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society The Boston Medical and Surgical Journal as published by EVOLUTION TOWARDS TWO MAIN TYPES OF HUMAI*
FIGURES,
DEPENDING
UPON
VARIATIONS
IÏ
THESE CORRELATIONS. struggle towards attaining competence of the
abdomen, with the consequent retention of the
viscera in position, in
a vertical trunk whose
dorsal and ventral surfaces tend constantly to
approach each other in approximately parallel
planes; this trunk being supported on two lower
extremities which are extended in the same ver-
tical plane, this extension being accompanied by
adduction and forward rotation,
so that the
patella and toes point forward. displays vigor
stability. On the whole, type (2), the retracted lower
thorax type, tends towards the development of
the following correlations: following
(a) Retarded or uncompleted competence of
the abdomen. f following
(a) Retarded or uncompleted competence of
the abdomen. f following
(a) Retarded or uncompleted competence of
the abdomen. (b) Predominance
of
forward expansion
of
the trunk during inspiration, forward and up-
ward, and forward and downward, with decreas-
ing inspiratory capacity and decreasing expira-
tory efficiency. patella
po t
This struggle may be tending, along the paths
indicated, towards the attainment of an equilib-
rium which makes for relative stability and the
release
of
energy
in
the form of specialized
activities of the various regions of the body. tory efficiency. (c) Less proportionate increase of transverse
over antero-posterior diameters, and tendency
to proportionate increase of antero-posterior over
transverse, especially in the abdomen. eg o s
body
Or, it may be tending, also along the paths indi-
cated,
towards compensations for failures
to
reach this relatively stable equilibrium. These
compensations result in types of figures which
may be merely unstable variants of the basic
generic type;
or the structural variations may be
so pronounced as to be called deformities. transverse, especially
(d) Pelvis tends to increased forward rotation. Lumbo-sacral anterior convexity tends to in-
crease. During weight bearing, lower extremi-
ties tend toward flexion at hip and knee, and feet
tend toward extension so that the plane of the
heel is higher than that of the forefoot. higher
(e) Trunk and extremities tend toward inter-
dependence. Trunk is in condition of unstable
equilibrium, and shares largely in the activities
of the extremities. There is retarded or unat-
tained independence
of the extremities;
the
activities of the extremities
are hampered by
the lack of the stable point of departure required
by voluntary muscles. EVOLUTION TOWARDS TWO MAIN TYPES OF HUMAI*
FIGURES,
DEPENDING
UPON
VARIATIONS
IÏ
THESE CORRELATIONS. types
gu e
These types are suggested in the higher mam-
malia, but they are fully developed in the human
being. being. The fundamental difference between these two
types appears in that portion of the trunk be-
tween the thoracic cavity and the heads of the
femora
— the
abdomen
and
pelvis. In
this
region the prominent points of differentiation
between the two types are the lower thorax; the
lumbo-sacral spine, including its sacral expan-
sion, the pelvis;
and the posterior, lateral and
anterior muscle walls of the abdomen and pelvis. ll d Further, this evidence seems to the writer to
justify the addition to the list of developmental
deformities of the added ones of round shoulders;
modifications of weak foot, including pes planus;
antero-posterior and lateral curvatures of the
spine; retracted lower thorax; marked lumbar
anterior convexity; marked forward rotation of
the pelvis; abdominal incompetence and visceral
ptosis
both en masse and individual. p
Broadly speaking, what may be called the basic
or primary type of figure is represented by a
broad lower thorax, a slight anterior convexity
of the lumbar spine, a slight anterior inclination
of the pelvis, and in selected cases, a competent
abdominal wall. The simplest variant of this
type has the abdomen incompetent but the back
unaffected. The next simplest variant shows
an incompetent abdomen, with hyperextension
of the lower thoracic spine, of the lumbo-thoracic
junction
or of the upper lumbar spine. ll d ptosis
—
These are developmental deformities because,
within physiological limits, there is a spontaneous
tendency to their occurrence in the efforts of
the individual to correlate the varying factors
enumerated. junction,
upper
spine. Again, broadly speaking, what may be called
the second type of figure is represented by a more
or less retracted lower thorax, a more pronounced
anterior convexity of the lumbar spine, extending
up into the thoracic spine, an increased anterior To sum up:
Th
f h
i di id
l To sum up:
Th
f p
The body of the individual at any given time
represents
a
moment
of equilibrium
in
the Hence, as this type is approached, the individual
displays vigor and stability. Hence, as this type is approached, the individual
displays vigor and stability. h l Hence, as this type is approached, the individual
displays vigor and stability. EVOLUTION TOWARDS TWO MAIN TYPES OF HUMAI*
FIGURES,
DEPENDING
UPON
VARIATIONS
IÏ
THESE CORRELATIONS. f p o ou ced
The existence of so many separate factors and
the number of natural laws simultaneously at
work make it impossible to trace the origin of
a given deformity in each case without having a
detailed life record of the individual. Many of
the deformities are, when first observed, of the
nature of cicatrices, their initial
cause having
disappeared in the presence of growth changes, or
being obscured by later growth changes. But
the further back into childhood one carries one's
studies and the greater the number of observa-
tions which one accumulates, the more the evi-
dence points to the existence of the fundamental
laws already formulated. by voluntary
(/) The
unnecessary expenditure
of
energy
following the foregoing decreases the power of
resistance of the individual, and favors a condi-
tion
of habitual overstrain which depreciates
powers of growth and development. Hence, as
this type is approached, the individual displays
weakness and instability. already
On the whole, type (1), the broad lower thorax
type,
tends towards the development of the
following correlations: CONCLUSIONS. Fundamental
cause of splanchnotosis is ab-
dominal incompetence. d
l
h
bd
i
l following
(a) Increasing competence of the abdomen. (b) P
d
i
f b
k
d
d
d following
(a) Increasing competence of the abdomen. (b) P
d
i
f b
k
d
d
d ( )
c eas g
p
(b) Predominance of backward and upward,
and backward and downward,
as well
as out-
ward expansion of the trunk, during inspiration,
with increasing inspiratory capacity and increas-
ing expiratory efficiency. p
In their highest development, the abdominal
walls not only contain the viscera, but also retain
them in position, against gravity and other dis-
placing forces. The abdomen may then be said
to be competent. Competence of the abdomen
is a developmental factor; stage of evolution with
which
human
race
is
at present struggling;
earlier phases can be traced backward through
vertebrate series and in human prenatal life. h
i i
f h
t ing expiratory efficiency. (c) Greater proportionate increase of trans-
verse
than
antero-posterior
diameters
of
trunk. (d) Pelvis maintains its affiliations with the
trunk. Lumbo-sacral anterior convexity tends
towards a minimum and the pelvis shows slight
anterior rotation. During weight-bearing, lower
extremities tend to maintain
extension at hip
and knee, and feet tend to maintain
such
an
angle of dorsal flexion as leads to ascent of the
forefoot to the horizontal plane of the heel. (d) Pelvis maintains its affiliations with the
trunk. Lumbo-sacral anterior convexity tends
towards a minimum and the pelvis shows slight
anterior rotation. During weight-bearing, lower
extremities tend to maintain
extension at hip
and knee, and feet tend to maintain
such
an
angle of dorsal flexion as leads to ascent of the
forefoot to the horizontal plane of the heel. p
Distinctive characteristic of human genus not
the erect trunk, nor even the erection of the trunk
on two hinder or lower extremities, but distinctive
human characteristic is the full extension of the
lower extremities in the plane of a vertical trunk,
the dorsal and ventral surfaces of which tend
constantly to approach each other in parallel
planes;
this extension being accompanied by
adduction and forward rotation, so that the pa-
tella and toes point forward. g
p
y
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by BY CHARLES GILMORE KERLEY, M.D., NEW YORK, Professor Diseases of Children, New York Polyclinic Medical School;
Attending Physician New York Infant Asylum; Attending Physi-
cian for Children, Sydenham Hospital; Assistant Attending Physi-
cian Babies' Hospital. The involuntary discharge of urine is normal
in the young infant. It becomes
a voluntary
function at a later age, the time depending largely
upon the child's training. In most children with
the right kind of management it may be con-
trolled during the waking hours by the tenth
month. During sleep it continues to
a later
period and while in many cases it may be under
perfect control, at the completion of the second
year I do not regard the loss of control as abnor-
mal until the third year is completed. If during
the second year the child shows a tendency to
frequent urination and involuntary passage of
urine during the waking hours with habitual
incontinence at night, it is my custom to advise
preventive measures. beyond. This instability of abdomen and lower extremi-
ties exists at the present day in the most primi-
tive as well as the most civilized peoples, and its
existence can be traced among all peoples as far
back as pictorial history extends. preventive
In some of these children the urine is very acid
and of a high specific gravity
— 1,020 or over. In such cases, a reduction of the highly nitrog-
enous food stuffs in the diet, especially meat and
eggs is often followed by improvement,
— the eggs
and red meat being given not offener than every
second day, alternately. Where the urine is
normal, the quantity of fluids given during the
twenty-four hours is reduced from 25% to 50%
and more solid nourishment substituted. pictorial history
Development of abdominal competence
also
correlated with postnatal evolution of abdominal
viscera. Evolution of viscera always incomplete
at birth;
considerable evidence to show that in
many cases never completed. many cases never completed. Development of spine, lower extremities, tho-
racic cage, and even upper extremities and head,
correlated with development of abdominal com-
petence,
and
vice
versa. Variations in
these
correlations result in spontaneous grouping of
body forms along two main paths:
(1) Basic or
primary type of figure, represented by broad lower
thorax, slight anterior convexity of lumbar spine,
slight anterior inclination of pelvis. BY CHARLES GILMORE KERLEY, M.D., NEW YORK, (2) Second-
ary or aberrant type of figure, represented by
retracted
lower thorax,
pronounced
anterior
convexity
of
lumbo-thoracic
spine,
increased
anterior inclination
of pelvis. Abdominal in-
competence and visceral ptosis in both types. Variations in the correlations enumerated show
a spontaneous
tendency
of
the
trunk
and
extremities towards the developmental deformi-
ties : round shoulders, lateral and antero-posterior
curvatures of the spine; modifications of weak
foot, including pes planus; retracted lower thorax,
marked lumbar or lumbo-thoracic anterior con-
vexity;
marked forward rotation of the pelvis;
abdominal incompetence, and visceral ptosis
—
both en masse and individual. di
i When the incontinence persists during the
waking hours at the completion of the second
year, or during sleep at the completion of the
third year, the condition is regarded as abnormal
and the child placed under treatment. placed
In assuming the care of a child with inconti-
nence our first step is to discover the cause of the
trouble. With this object in view the patient is
examined with the idea of discovering any periph-
eral abnormality
which
may
have
a bearing
on the disorder. Thus the incontinence may be
due to a vaginitis, or to an adherent clitoris in
girls, or to phimosis in boys; it may be due to
thread-worms in the rectum, to constipation, to
stone in the bladder, to cystitis,
—
a very rare
condition,
— and to hyperacidity of the urine,
—
a very common one. The diet may play a part. The
use of highly nitrogenous food in large
amounts, or
a diet rich in sugar may lead to
changes in the urine sufficient to cause the trouble. The presence of adenoid growths in the naso-
pharyngeal vault is supposed by
some writers
to cause enuresis. Experiments
and
dissections
support
the
clinical hypothesis that abdominal incompetence
is the primary factor in the development not
only of splanchnoptosis, but also of the common
surgical diseases of the
upper abdomen, the
stomach, duodenum, gall bladder and bile ducts,
jejuno-ileum,
pancreas and kidneys (especially
the right) being
the most important
viscera
affected. As a result of the diurnal and nocturnal incon-
tinence, the bladder may never have developed
and its capacity may be greatly reduced. Obvi-
ously, when such is the case, incontinence will
be noted both day and night. day
night. After all possible dietetic and peripheral causes
have been eliminated, about 90% of the cases
remain. CONCLUSIONS. plane
(e) Independence of action of trunk and ex-
tremities; trunk tends to such stability that it
furnishes only a point of departure for develop-
ing the activities of the extremities, with a mini-
mum participation in such activities;
extremi-
ties tend to develop such independence of action
that their call upon the trunk is minimized. point
Development of abdominal competence
cor-
related with evolution of this extension, adduction
and forward rotation of the lower extremities. At birth, abdomen is incompetent, and lower
extremities (thighs)
are flexed, abducted and
rotated outward. Postnatal evolution of lower p
(/) The conservation of energy following the
foregoing increases the power of resistance of the
individual, and favors growth and development. extremities in direction of extension, adduction
and forward rotation tends to be completed in
early childhood, but is an unstable possession,
and in a large number of cases never fully at-
tained; in another large number of cases is at-
tained but after varying periods of time, more
or less reversion to the condition found at birth
occurs; tendency to this reversion always present,
but becomes
more pronounced with approach
of fourth decade and beyond. Postnatal
evo-
lution of abdomen in direction of competence
proceeds throughout infancy and childhood, and,
in selected cases, is complete at or before puberty. Very unstable possession;
in majority of cases
never fully attained;
even when attained, ten-
dency to reversion to condition of more or less
incompetence constantly present; tendency be-
comes more pronounced with approach of fourth
decade and beyond. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on June 22, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society INCONTINENCE OF THE URINE. extremities in direction of extension, adduction
and forward rotation tends to be completed in
early childhood, but is an unstable possession,
and in a large number of cases never fully at-
tained; in another large number of cases is at-
tained but after varying periods of time, more
or less reversion to the condition found at birth
occurs; tendency to this reversion always present,
but becomes
more pronounced with approach
of fourth decade and beyond. Postnatal
evo-
lution of abdomen in direction of competence
proceeds throughout infancy and childhood, and,
in selected cases, is complete at or before puberty. Very unstable possession;
in majority of cases
never fully attained;
even when attained, ten-
dency to reversion to condition of more or less
incompetence constantly present; tendency be-
comes more pronounced with approach of fourth
decade and beyond. BY CHARLES GILMORE KERLEY, M.D., NEW YORK, The Boston Medical and Surgical Journal as published by
l
f M di i
D
l
d d f
j
SAN DIEGO (UCSD)
J
22 2016 BY CHARLES GILMORE KERLEY, M.D., NEW YORK, These are due to a neurosis, and are not
dependent upon any discoverable pathological
condition. There is
a lack of development,
a
weakness of the vesical sphincter and a lack of The Chicago Isolation Hospital has had
no patients
since July 18, when the last of the 19 smallpox patients
discovered since Jan. 1 was discharged.— Journal A.M.A.
|
https://openalex.org/W4382290665
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https://magister.wisnuwardhana.ac.id/index.php/Perspektif/article/download/54/46
|
Indonesian
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UPAYA PENCEGAHAN FRAUD PADA BANK BERPLAT MERAH YANG MERUGIKAN KEUANGAN NEGARA
|
Jurnal Magister Hukum Perspektif
| 2,022
|
cc-by-sa
| 5,455
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Abstrak Fraud bisa terjadi dimana saja dan kapan saja termasuk pada bank berplat merah
(Bank Milik Negara). Sebagai tempat perputaran uang, perbankan tidak lepas dari
tindak pidana baik tindak pidana umum, perdata, dan tindak pidana korupsi oleh
karena Bank memiliki kedudukan yang rentan terhadap penyalahgunaan
kewenangan baik oleh pegawai pada lini depan seperti teller, costumer service, loan
service atau oleh Kepala Cabang bahkan pada jajaran Direksi. Rumusan masalah
yang diangkat adalah bagaimana bentuk upaya pencegahan fraud khususnya pada
Bank Milik Negara yang berkaitan dengan penyelamatan aset dan kekayaan negara
yang terindikasi merugikan keuangan negara. Jenis penelitian yang digunakan
adalah yuridis-normatif yaitu salah satu metode penelitian hukum yang diperoleh
dengan menganalisis suatu permasalahan hukum melalui peraturan perundang-
undangan dan literatur terkait. Hasil yang ditemukan upaya pencegahan terdiri dari
aspek teknis dan moral serta integritas. Aspek teknis dapat melalui (1) Cek ulang
data nasabah, (2) Memberikan Reward Kepada Penemu Celah, (3) Ciptakan kontrol
internal yang baik, (4) Transparansi kepada Nasabah, (5) Tingkatkan Pengawasan
Personil, (6) Terapkan e-KYC, (7) Whistleblower. Aspek moral dan integritas harus
dimiliki oleh setiap pihak yang terlibat dalam kegiatan di bank. Selain itu, Aparat
Penegak Hukum (APH) juga harus mematuhi penerapan strategi anti-fraud bank
umum yang telah diatur dalam POJK guna Penyelamatan Keuangan Negara dan
Aset Negara serta Pemulihan Ekonomi Nasional. K
k
i
h
f
d b
k Kata kunci: Pencegahan, fraud, bank. 1 Alamat Korespondensi: artupvoir@gmail.com UPAYA PENCEGAHAN FRAUD PADA BANK BERPLAT MERAH
YANG MERUGIKAN KEUANGAN NEGARA Rio Vernika Putra1
Mahasiswa Magister Ilmu Hukum Universitas Wisnuwardhana Malang Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 14 Implementing e-KYC, (7) Whistleblowers. Moral and integrity aspects must be
owned by every party involved in activities in the bank. In addition, Law
Enforcement Officials (APH) must also comply with the implementation of the
commercial bank anti-fraud strategy that has been regulated in the POJK for the
Rescue of State Finances and State Assets and National Economic Recovery. Keywords: Prevention, fraud, bank. Abstract Fraud can occur anywhere and anytime including in red-plated banks (State-
Owned Banks). As a place of money turnover, banking cannot be separated from
criminal acts both general, civil, and corruption crimes because the Bank has a
position that is vulnerable to abuse of authority either by employees on the front
line such as tellers, costumer services, loan services or by branch heads even on
the Board of Directors. The formulation of the issue raised is how the form of fraud
prevention efforts, especially in State-Owned Banks related to the rescue of assets
and wealth, is indicated to be detrimental to the country's finances. The type of
research used is juridical-normative which is one of the legal research methods
obtained by analyzing a legal problem through laws and regulations and related
literature. The results found prevention efforts consist of technical and moral
aspects and integrity. Technical aspects can be through (1) Double-check customer
data, (2) Rewarding Loophole Inventors, (3) Creating good internal controls, (4)
Transparency to Customers, (5) Increasing Personnel Supervision, (6) 14
Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 2 ACFE, 2020, Report To The Nations 2020 Global Study on Occupational Fraud and Abuse,
US, Association of Certified Fraud Examiners, hlm. 8.
3 Muhammad Choirul Anwar, 2020, “ Tersangka Kasus Suap Miliaran, Eks Dirut BTN Ditahan
Kejagung”, https://www.cnbcindonesia.com/market/20201010141502-17-193389/tersangka-kasus-
suap-miliaran-eks-dirut-btn-ditahan-kejagung. Diakses pada tanggal 2 Oktober 2020. A. Latar Belakang Kejahatan perbankan ada banyak macamnya, salah satunya adalah fraud
atau kecurangan yang terjadi pada perbankan dimana bank bisa menjadi korban dan
bisa juga bank sebagai sarana bagi pegawai bank untuk menjadi pelaku dari fraud
pada perbankan tersebut. Banyaknya kegiatan perbankan yang rawan dan rentan
terhadap penyalahgunaan kewenangan (fraud) oleh pihak bank menuntut bank agar
selalu menerapkan prinsip kehati-hatian yang didalamnya terkandung sifat untuk
mematuhi peraturan yang ada serta cara paling efisien dan efektif untuk
menyelesaikan fraud (best practice) pada sektor perbankan. Dalam perbankan fraud atau kecurangan merupakan suatu tindakan
penyimpangan yang bertentangan dengan hukum dan dengan sengaja melanggar
ketentuan internal organisasi yang meliputi sistem, kebijakan, dan juga prosedur
yang berpotensi merugikan bank baik secara materiil maupun secara moril. Dalam
industri perbankan. Fraud atau kecurangan dapat diartikan pula sebagai suatu
tindakan atau perbuatan yang dengan maksud disengaja menggunakan sumber daya
organisasi atau perusahaan secara tidak wajar untuk memperoleh keuntungan
pribadi sehingga merugikan pihak organisasi atau perusahaan yang bersangkutan
atau pihak lain. Dalam peraturan OJK (otoritas jasa keuangan) Nomor 39 tahun
2019 tentang Penerapan Strategi Anti-Fraud bagi Bank Umum disebutkan bahwa
fraud adalah tindakan penyimpangan atau pembiaran yang sengaja dilakukan untuk
mengelabuhi, menipu, atau memanipulasi bank, nasabah, atau pihak lain, yang
terjadi di lingkungan bank dan/atau menggunakan sarana bank sehingga
mengakibatkan bank, nasabah, atau pihak lain menderita kerugian dan/atau pelaku
fraud memperoleh keuntungan keuangan baik secara langsung maupun tidak
langsung. Sampai dengan saat ini kasus fraud perbankan terus terjadi meskipun telah
banyak pelaku fraud yang telah dijatuhi hukuman sesuai dengan ketentuan hukum
yang berlaku. Akan tetapi kasus fraud terus terjadi dan semakin bervariasi bahkan
ada beberapa kasus fraud yang telah merugikan keuangan negara artinya hingga
saat ini masih memerlukan strategi pencegahan fraud di perbankan oleh Aparat
Penegak Hukum (APH) dan pihak Perbankan khususnya Bank Milik Negara
sehingga kepastian perlindungan bank kepada nasabah dan terciptanya sistem
deteksi dini (early warning system) dapat optimal. Rumusan masalah yang akan diangkat adalah bagaimana bentuk upaya
pencegahan fraud khususnya pada Bank Milik Negara yang berkaitan dengan
penyelamatan aset dan kekayaan Negara yang terindikasi merugikan keuangan
negara. A. Latar Belakang Jenis penelitian yang digunakan adalah yuridis-normatif yaitu salah satu
metode penelitian hukum yang diperoleh dari studi kepustakaan dengan
menganalisis suatu permasalahan hukum melalui peraturan perundang-undangan, Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 15 Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 15 Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 15 literatur terkait, dan bahan referensi lainnya yang berhubungan dengan pencegahan
fraud pada bank berplat merah yang mengakibatkan kerugian keuangan negara. 4 Gamlath Mohottige, et.all. 2018, “The New Fraud Triangle Theory - Integrating Ethical
Values Of Employees”, International Journal of Business, Economics and Law, Vol. 16, Issue 5
(August), p. 52-57. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 16 menerapkan strategi anti-fraud secara efektif. Penyebab fraud pada Bank Milik
Negara menurut Himpunan Bank Milik Negara (HIMBARA) yang pertama adalah
karena adanya niat dari pelaku fraud dan yang kedua adanya kesempatan untuk
melakukan fraud. Kriminolog Donald Ray Cressey dalam teori fraud triangle menyatakan
bahwa sebuah kejahatan timbul karena adanya 3 hal yaitu4 Kriminolog Donald Ray Cressey dalam teori fraud triangle menyatakan
4 o og
onald
ay C essey da a
teo
f aud
bahwa sebuah kejahatan timbul karena adanya 3 hal yaitu4 bahwa sebuah kejahatan timbul karena adanya 3 hal yaitu4 1. Tekanan, seperti yang diilustrasikan oleh Cressey Donald pada tahun 1953,
adalah insentif yang dapat memotivasi seseorang untuk terlibat dalam
penipuan. Tekanan dapat dihasilkan dari masalah pribadi, seperti tekanan
keuangan atau tekanan kecanduan, atau dari lingkungan kerja. Dorongan yang
timbul dalam diri seseorang yang dijadikan alasan untuk melakukan kejahatan. Alasan motivasi ini dibagi menjadi dua bagian yaitu alasan financial dan alasan
non financial. Agar menjadi lebih jelas berikut beberapa contoh alasan
financial antara lain : a. Terlilit hutang; b. Kebutuhan belanja yang besar untuk hutang dan modal; c. Gaji yang rendah; d. Insentif yang kecil; e. Kehilangan pekerjaan; f. Pengambilan saham dari pemegang saham yang bukan pengendali; g. Market expectation yang terlalu tinggi; h. Terlibat perbuatan kejahatan. Sedangkan untuk alasan non financial antara lain sebagai berikut : a. Gaya hidup yang tidak sesuai dengan besaran gaji; b. Sifat rakus/tamak; c. Tekanan sosial; d. Tekanan atasan; e. Tidak mampu mengikuti perubahan teknologi; f. Adanya regulasi baru; g. Ketidak mampuan memenuhi syarat yang sudah ditetapkan pemerintah; h. Kurang disiplin; h. Kurang disiplin; i. Tidak berintegritas. B. Pembahasan Beberapa kasus fraud yang terungkap di sektor perbankan Indonesia
hingga saat ini masih dapat dijumpai. Hal tersebut menunjukkan bahwa pelaku
fraud hampir ada disetiap bank termasuk didalam bank milik negara. Association
of Certified Fraud Examiners (ACFE) di bulan Agustus 2020 yang merupakan
organisasi terbesar anti-fraud di level global merilis Report To The Nations
(RTTN). RTTN mengungkapkan bahwa tercatat 2.504 kasus fraud dari 125 negara2
dengan median loss USD 8.300 per bulan, dan terhitung ada 29 kasus fraud di
Indonesia. Kasus yang menonjol adalah pada bulan Oktober 2020 lalu, Mantan
Direktur Utama Bank BTN, Maryono ditangkap oleh Kejaksaan Agung atas dugaan
menerima gratifikasi dari debitur sebanyak 2 kali yaitu sejumlah Rp. 2,257 miliar
dan Rp. 870 juta yang ditransfer ke rekening menantunya3. Ini artinya peristiwa
fraud bisa terjadi dimana saja dan oleh siapa saja baik itu pegawai pada lini depan
(Teller, Custumer Service, Loan Service), Kepala Cabang, sampai ke jajaran
Direksi. Dalam Forum Koordinasi antara Himpunan Bank Milik Negara (Himbara)
yaitu Bank Mandiri, BNI, BRI dan BTN dengan Aparat Penegak Hukum (APH)
yang digelar di Press Room Kejaksaan Agung Kebayoran Jakarta Selatan, dalam
forum koordinasi tersebut perwakilan dari Himpunan Bank Milik Negara
(HIMBARA) menyampaikan sesuai dengan Peraturan OJK Nomor 39 Tahun 2019
Tentang Penerapan Strategi Anti-Fraud bagi Bank Umum, disebutkan bahwa Fraud
adalah tindakan penyimpangan atau pembiaran yang sengaja dilakukan untuk
mengelabui, menipu, atau memanipulasi Bank, nasabah, atau pihak lain, yang
terjadi di lingkungan Bank dan/atau menggunakan sarana Bank, sehingga
mengakibatkan Bank, nasabah, atau pihak lain menderita kerugian dan/atau pelaku
fraud memperoleh keuntungan keuangan baik secara langsung maupun tidak
langsung. Menurut Himpunan Bank Milik Negara (HIMBARA), dalam perbankan,
pengawasan untuk mencegah terjadinya kecurangan (fraud) menjadi salah satu
fokus utama yang paling dijaga. Otoritas Jasa Keuangan (OJK) sebagai regulator
dan pengawas lembaga keuangan termasuk Bank-pun telah melakukan evaluasi
sekaligus memperketat aturan di perbankan agar ruang terjadinya fraud semakin
sempit. Sesuai ketentuan mengenai manajemen risiko, Bank diwajibkan memiliki
kebijakan dan prosedur untuk mengelola risiko, termasuk adanya sistem
pengendalian intern terhadap pelaksanaan kegiatan usaha dan operasional pada
seluruh jenjang organisasi Bank Selanjutnya perwakilan dari Himpunan Bank Milik
Negara (HIMBARA) menyampaikan, pengaturan mengenai pencegahan fraud di
industri perbankan telah berlaku sejak tahun 2011, dan terakhir disempurnakan
pada POJK No.39/POJK.03/2019 tentang Penerapan Strategi Anti-Fraud. Melalui
POJK 39/2019 tersebut, regulator mewajibkan Bank untuk menyusun dan 16
Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 2. Kesempatan Tekanan menciptakan motif kejahatan yang akan dilakukan, tetapi
karyawan juga harus memahami bahwa ia memiliki kesempatan untuk
melakukan kejahatan tanpa tertangkap. Dalam pandangan Cressy, ada dua
komponen dari kesempatan yang dirasakan untuk melakukan pelanggaran
kepercayaan; informasi umum dan keterampilan teknis. Informasi umum
hanyalah pengetahuan bahwa posisi kepercayaan karyawan dapat dilanggar. Keterampilan teknis mengacu pada kemampuan yang dibutuhkan untuk
melakukan pelanggaran. Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 17 Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 17 Kesempatan ini dianggap sebagai solusi atas alasan-alasan financial dan
alasan non financial sebagaimana tersebut diatas. Kesempatan ini merupakan
keadaan yang sesungguhnya terjadi di lapangan seperti : a. Lemahnya pengawasan internal; b. Relasi yang intens dengan konsumen; c. Kesulitan menilai kualitas kinerja karyawan; d. Ketidakmampuan untuk mendeteksi adanya fraud; e. Kepercayaan nasabah yang tinggi terhadap agennya; f. Tidak adanya skimming detection. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 18 Yang pertama Skimming adalah tindakan mengambil data nasabah
menggunakan alat perekam yang biasanya kejahatan cyber ini terjadi di mesin
elektronic data capture (EDC) dan juga dimesin anjungan tunai mandiri atau ATM. Kedua Phising yaitu modus para penjahat dengan membuat web yang serupa
dengan web transaksi perbankan dengan pengamanan hanya menggunakan user id
dan password sehingga para pelaku dengan mudah dapat mengambil dana yang ada
direkening nasabah. Saat ini phising lebih canggih lagi karena bisa menyerang
komputer nasabah dan web perbankan terutama transaksi yang menggunakan
internet banking, dengan pengamanan multi faktor saja masih dapat ditembus oleh
para pelaku fraud. Sedangkan yang ketiga adalah Malware. Malware (Virus,
Worm, Trojan, dan ancaman lainnya)5 adalah ancaman paling penting yang tersedia
dari penjahat cyber untuk mendapatkan akses tidak sah ke akun pengguna untuk
mencuri data keuangan mereka dan informasi sensitif lainnya. Pertumbuhan yang
cepat dalam perangkat mobile seperti Smartphone dan Tablet PC mengarah pada
pengembangan lebih lanjut dari perangkat lunak berbahaya malware. Hacking &
Cracking: Melalui hacking dan cracking penipu komputer dapat masuk ke
komputer dan jaringan komputer untuk mencuri informasi keuangan yang nantinya
dapat digunakan untuk tujuan yang tidak sah. Perangkat lunak berbahaya yang
berbeda dapat digunakan untuk tujuan hacking oleh penipu komputer seperti virus
Trojan, kerangka kegiatan rutin untuk mengeksplorasi kehilangan data yang
disebabkan oleh infeksi malware. Berdasarkan gambaran diatas, dapat diketahui bahwa kejahatan perbankan
yang terjadi di bank berplat merah sangat bervariasi. Jika hal ini tidak segera
diperbaiki maka dalam jangka panjang akan memepengaruhi stabilitas keuangan
nasional, selain itu dapat diketahui bahwa para pelaku fraud di Bank Milik Negara
justru berada di level manajerial dengan tekanan financial sebagai motivasi utama,
ini menunjukkan bahwa kekuasaan yang diberikan kepada level manajerial tidak
dibarengi dengan pengawasan yang ketat atas setiap tindakan dan kebijakan yang
dilakukan. Beberapa modus operandi fraud yang dilakukan oleh pelaku fraud dilevel
manajerial adalah dibidang kredit seperti adanya kredit macet yang disebabkan oleh
lemahnya pengawasan internal serta aturan kredit yang tidak di update atau
diperbarui sesuai dengan perkembangan jaman, bidang pendanaan, bidang
operasional seperti penyalahgunaan bilyet giro dan deposito. 5 Vijayalakshmi P, et.all, 2021, “Impacts Of Cyber Crime On Internet Banking”, International
Journal of Engineering Technology and Management Sciences, Issue: 2 Volume No.5 March – 2021,
p. 30-34. 3. Rasionalisasi Cressey menunjukkan bahwa rasionalisasi bukanlah cara ex post facto
untuk membenarkan pencurian yang telah terjadi. Secara signifikan,
rasionalisasi adalah komponen penting dari kejahatan sebelum terjadi; bahkan,
itu adalah bagian dari motivasi untuk melakukan kejahatan, karena
penggelapan tidak melihat dirinya sebagai penjahat, dia harus membenarkan
kesalahannya sebelum dia melakukannya. Rasionalisasi diperlukan agar
pelaku, dapat membuat perilaku ilegalnya dapat dimengerti oleh dirinya
sendiri dan mempertahankan konsep dirinya sebagai orang yang dipercaya. Adapun bentuk-bentuk dari rasionalisasi adalah sebagai berikut : a. Merasa berjasa pada perbankan tempat yang bersangkutan bekerja
sehingga dapat mengambil keputusan sepihak; b. Melihat rekan kerja atau atasan melakukan fraud akan tetapi tidak
dipermasalahkan; c. Manajemen tidak segera memperbaiki kelemahan sistem yang telah
diketahui; d. Manajemen mengerjakan hal yang bukan menjadi tanggung jawabnya; e. Perbankan tidak membedakan antara transaksi bisnis dan transaksi
personal; f. Adanya sengketa pada pemegang saham; g. Adanya alasan non material atas standart akuntansi yang tidak sesuai; h. Adanya kepentingan manajemen yang mendasarkan pada kedudukan atau
jabatannya untuk mengambil keputusan; i. Standart etika manajemen yang tidak efektif sehingga tidak perlu
implementasi nilai-nilai dalam entitas; j. Pegawai yang pernah melanggar ketentuan perundangan kemudian
ditempatkan pada posisi yang sama dan melakukan pekerjaan yang sama
kembali. Selain ketiga unsur tersebut (motivasi, kesempatan, dan rasionalisasi)
terdapat fraud yang dilakukan dengan keahlian khusus yang tidak dimiliki oleh
setiap orang yang lebih dikenal dengan istilah cybercrime banking fraud yang
dilakukan dengan cara memanfaatkan kelemahan sistem perusahaan dan kelemahan
manajemen. Perwakilan Himpunan Bank Milik Negara (HIMBARA) yang terdiri
dari Bank Mandiri, BNI, BRI dan BTN mengatakan setidaknya ada 3 (tiga) modus
dalam kejahatan cyber yang menyerang sistem perbankan Indonesia yaitu
Skimming, Phising, dan Malware. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Terjadinya
pembobolan bank secara manual atau melalui sarana elektronik seperti internet
dapat terjadi karena lemahnya sistem perbankan serta pengawasan atas laporan
keuangan perbankan dan nasabah yang tidak efektif menjadi kasus yang paling
sering terjadi dan yang terakhir mengenai pembenaran tindakan kejahatan
perbankan yang dilakukan karena standart etika manajemen tidak dilaksanakan
secara terus menerus dan adanya asumsi bahwa kerugian tidak bersifat material
serta mempunyai sifat yang merasa paling berjasa bagi perbankan harus
dihilangkan. Tidak dapat dipungkiri bahwa kejahatan selalu lebih dulu ada Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 19 Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 19 dibandingkan dengan kebijakan, hal ini dapat diketahui Bank Indonesia beberapa
kali mengeluarkan kebijakan setelah kejahatan terjadi dimana pelaku berdalih
bahwa belum ada peraturan yang mengatur tentang hal tersebut. p
y
g
g
g
Berdasarkan analisa diatas dapat diperoleh beberapa indikasi perbankan
yang mengalami fraud antara lain sebagai berikut : Berdasarkan analisa diatas dapat diperoleh beberapa indikasi perbankan
yang mengalami fraud antara lain sebagai berikut : a. Akun lama nasabah tiba-tiba aktif kembali; b. Tanda tangan nasabah yang tidak konsisten dan nasabah sulit untuk dihubungi;
h
k i i
k di d l
j
l h
id k
j b. Tanda tangan nasabah yang tidak konsisten dan nasabah sulit untuk dihubungi;
c. Permohonan atau aktivitas kredit dalam jumlah yang tidak wajar; g
y
g
g ;
c. Permohonan atau aktivitas kredit dalam jumlah yang tidak wajar; d. Data nasabah yang berubah, tidak konsisten, tampak seperti dimanipulasi, ID
Card tidak jelas dan ada riwayat atau catatan dari BI checking; e. Sistem perbankan yang tidak terpakai sebelumnya dan digunakan kembali
untuk alasan tertentu; f. Nasabah tidak dapat memberikan jawaban seperti pada data perbankan; g. Surat yang dikirimkan kepada nasabah dikembalikan secara terus menerus,
padahal pada catatan bank, nasabah tersebut masih aktif. 1. Strategi pencegahan fraud pada Bank Milik Negara g p
g
f
p
g
Belum optimalnya kepastian perlindungan bank kepada nasabah dan
belum adanya sistem informasi tentang sistem deteksi dini (early warning system),
serta diperlukannya pemahaman yang sama antar Aparat Penegak Hukum (APH)
dengan pihak Perbankan (khususnya Bank Milik Negara) mengenai strategi
pencegahan fraud di Perbankan. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 20 Sebelum membahas mengenai strategi pencegahan fraud pada Bank Milik
Negara kami mengingatkan kembali bahwa Fraud adalah tindakan yang sengaja
dilakukan untuk menipu dan mengelabui Bank, nasabah atau pihak lain, yang
mengakibatkan kerugian pada pihak yang dikelabui dan keuntungan bagi pelaku
fraud baik secara langsung maupun tidak langsung. Fraud yang sering terjadi pada
dunia perbankan umumnya terjadi pada dua sistem yakni sistem Digital
Banking dan Pemberian Kredit. Berbagai Bank terus berupaya untuk mencegah
fraud dan terus mensosialisasikan bagaimana cara untuk mencegah terjadinya fraud
berikut beberapa cara yang dapat digunakan sebagai peringatan dini dan dapat
diterapkan untuk mencegah fraud di Bank Milik Negara, sebagai berikut : 1. Cek Ulang Data Nasabah
Proses ini bertujuan untuk mengantisipasi adanya nasabah yang mempunyai
niat tidak baik dan berencana bekerjasama oknum pada bank untuk
memanipulasi data laporan keuangan. Selain nasabah, data laporan
keuangan supplier juga sebaiknya dilakukan cek ulang. 2. Memberikan Reward Kepada Penemu Celah
Hal ini guna mengantisipasi fraud pada sistem digital banking, selama ini
peretas banyak terkesan negatif. Namun siapa sangka ternyata di Indonesia ini
ada orang-orang yang memiliki profesi sebagai ethical hacker yang dapat
ditugaskan untuk mencari bug dan celah keamanan kemudian mampu
memperbaikinya. Jika pihak bank mendapatkan laporan adanya bug dan celah
yang tidak disadari oleh pihak IT internal bank itu sendiri, mintalah pelapor
untuk
mendemokan
bagaimana
celah
itu
ditemukan,
kemudian
berikanlah rewards kepada mereka karena telah membantu bank untuk
mengamankan sistemnya dari fraud. Karena jika bank bersikap cuek kepada
mereka yang telah berusaha membantu pihak bank, bisa jadi mereka malah
menyebarkan bagaimana cara mereka meretas bank di forum-forum berisi
peretas, bahkan menyebarkan data penting nasabah. Pada sistem digital, vendor
pihak eksternal yang memiliki tenaga ahli di bidang ini memang cukup
dibutuhkan perannya untuk membantu mengawasi adanya bug atau celah dan
hal itu berguna untuk mencegah fraud pada sistem digital perbankan. 3. 3. Ciptakan kontrol internal yang baik
Kontrol internal yang baik paling tidak harus mencakup kontrol lingkungan,
sistem akuntansi, dan kontrol prosedur (aktivitas), merujuk pada
pernyataan Committee of Sponsoring Organization (COSO): Lingkungan
pengendalian menentukan irama organisasi,sebagian besar bertanggung jawab
untuk membuat memberi kesadaran terhadap karyawan sehingga dapat
waspada terhadap segala kontrol. Yang perlu ditekankan disini bahwa tidak ada
sistem kontrol internal yang kebal terhadap fraud. Efektifitas akan sangat
bergantung pada kompetensi orang-orang di bank yang harus memastikan
pelaksanaan kontrol internal yang tepat. Sistem kontrol internal hanyalah salah
satu elemen program pencegahan fraud yang komprehensif. 4. 4. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Adanya persamaan persepsi dengan cara
membangun sebuah kolaborasi lintas sektor antara Aparat Penegak Hukum yaitu
Kejaksaan Agung dengan Himbara (Himpunan Bank Milik Negara) yang terdiri
dari Bank Mandiri, BRI, BNI, dan BTN dalam jangka pendek serta dapat
menggandeng OJK (Otoritas Jasa Keuangan) jangka menengah dan diharapkan
jangka Panjang kolaborasi ini akan diperkuat dengan Aparat Penegak Hukum
(APH) lainnya yaitu Kepolisian dan Komisi Pemberantasan Korupsi (KPK) serta
Pusat Pelaporan dan Analisis Transaksi Keuangan (PPATK), Bank Indonesia,
Kementerian Keuangan, Lembaga Penjamin Simpanan, Forum Koordinasi
Stabilitas Sistem Keuangan, dan stakeholders lainnya, dimana tujuan proyek
perubahan melalui inovasi dan integrasi dalam bentuk kolaborasi lintas sektoral
pencegahan fraud ini akan bermanfaat untuk memperkuat sistem anti-fraud pada
Bank Milik Negara khususnya dalam pilar pencegahan, penguatan peringatan dini
(early warning system) yang lebih cepat, efektif, valid, dan komprehensif. Lalu
terciptanya Whole of Government (WoG) di antara para penegak hukum dalam
rangka Pencegahan tindakan Fraud di Bank Milik Negara yang holistik, akurat, dan
sistematis dalam penyelamatan aset dan kekayaan negara serta mewujudkan Good
Coporate Governance dan pada akhirnya adanya kepastian dan perlindungan bagi
Bank dan Nasabah, serta tercipta zero fraud. Forum koordinasi antara Aparat
Penegak Hukum (APH) dengan Himbara (Himpunan Bank Milik Negara) bertujuan
untuk menciptakan kolaborasi ekosistem ekonomi dengan ekosistem Aparat
Penegak Hukum (APH) oleh karena tindakan pencegahan tidak dapat dilakukan
sendiri-sendiri karena fraud yang terjadi merupakan dampak dari ekosistem yang
jika tidak kolaborasi akan sulit sekali ditangani. Himbara (Himpunan Bank Milik
Negara) juga meminta adanya penguatan sistem deteksi dini (early warning system)
untuk memperkuat tindakan pencegahan. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Transparansi kepada Nasabah
Pada awal penerimaan nasabah dijelaskan mengenai hak dan kewajiban
nasabah maupun pihak bank sebagai Contoh termudahnya mengenai perilaku
suap dari nasabah guna mendapatkan kucuran dana dari bank melalui oknum Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 21 Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 21 di bank tersebut. Disini bank bisa membuat surat secara periodik kepada
nasabah terkait, guna menjelaskan mengenai kebijakan perusahaan yang tidak
menerima segala jenis suap atau hadiah. Hal yang juga diharapkan dapat
meminimalisir niat nasabah untuk melakukan fraud adalah bahwa bank harus
menginformasikan diawalan kebijakan bahwa bank memiliki hak akses yang
bisa digunakan sewaktu-waktu untuk mengaudit laporan keuangan nasabah
yang memperoleh pinjaman tersebut 5. 5. Tingkatkan Pengawasan Personil
Pengawasan terhadap personil secara periodik sangat diperlukan guna
mencegah fraud oleh oknum perbankan. Pelaku fraud biasanya menggunakan
hasil jarahannya untuk mendukung gaya hidup yang mahal. Dengan
mengawasi gaya hidup setiap personil dan fasilitas-fasilitas pribadi di
sekelilingnya, bank bisa melakukan langkah pencegahan. Sebab para personil
yang berpotensi melakukan fraud seakan-seakan merasakan terus diawasi
sehingga pencegahan akan terjadinya fraud dapat dicegah lebih efektif. 5. Tingkatkan Pengawasan Personil
Pengawasan terhadap personil secara periodik sangat diperlukan guna
mencegah fraud oleh oknum perbankan. Pelaku fraud biasanya menggunakan
hasil jarahannya untuk mendukung gaya hidup yang mahal. Dengan
mengawasi gaya hidup setiap personil dan fasilitas-fasilitas pribadi di
sekelilingnya, bank bisa melakukan langkah pencegahan. Sebab para personil
yang berpotensi melakukan fraud seakan-seakan merasakan terus diawasi
sehingga pencegahan akan terjadinya fraud dapat dicegah lebih efektif. 6. 6. Menerapkan Electronic Know Your Custumer (eKYC)
Electronic Know Your Custumer (eKYC) adalah proses pengenalan calon
nasabah dengan memanfaatkan peran teknologi. Proses eKYC ini dinilai lebih
baik dari segi waktu dan keamanan dibanding proses KYC manual yang
biasanya dilakukan oleh Bank. Bahkan Otoritas Jasa Keuangan (OJK) terus
mendorong industri keuangan agar menerapkan eKYC pada proses bisnis
mereka, sesuai dengan Peraturan Otoritas Jasa Keuangan (OJK) Nomor
23/POJK.01/2019 tentang Penerapan Program Anti Pencucian Uang dan
Pencegahan Pendanaan Terorisme di Sektor Jasa Keuangan. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 22 Kerja
Karyawan. Bank
juga
telah
membentuk
Petugas
Fungsi
Anti Fraud berdasarkan POJK No.39/POJK.03/2019, tentang Penerapan Strategi
Anti Fraud bagi Bank Umum. Petugas Fungsi Anti Fraud merupakan fungsi yang
bertugas menangani penerapan strategi Anti Fraud dalam organisasi Bank. Petugas
Fungsi Anti Fraud terdiri dari : Kerja
Karyawan. Bank
juga
telah
membentuk
Petugas
Fungsi
Anti Fraud berdasarkan POJK No.39/POJK.03/2019, tentang Penerapan Strategi
Anti Fraud bagi Bank Umum. Petugas Fungsi Anti Fraud merupakan fungsi yang
bertugas menangani penerapan strategi Anti Fraud dalam organisasi Bank. Petugas
Fungsi Anti Fraud terdiri dari : Kerja
Karyawan. Bank
juga
telah
membentuk
Petugas
Fungsi
Anti Fraud berdasarkan POJK No.39/POJK.03/2019, tentang Penerapan Strategi
Anti Fraud bagi Bank Umum. Petugas Fungsi Anti Fraud merupakan fungsi yang
bertugas menangani penerapan strategi Anti Fraud dalam organisasi Bank. Petugas
Fungsi Anti Fraud terdiri dari : Kerja
Karyawan. Bank
juga
telah
membentuk
Petugas
Fungsi
Anti Fraud berdasarkan POJK No.39/POJK.03/2019, tentang Penerapan Strategi Anti Fraud bagi Bank Umum. Petugas Fungsi Anti Fraud merupakan fungsi yang
bertugas menangani penerapan strategi Anti Fraud dalam organisasi Bank. Petugas
Fungsi Anti Fraud terdiri dari : 1. Direktur Yang Membawahi Fungsi Kepatuhan sebagai Koordinator; 2. Kepala Divisi Kepatuhan dan APU PPT, sebagai anggota; 3. Kepala Divisi Sumber Daya Manusia sebagai anggota; ivisi Sumber Daya Manusia sebagai anggota 4. Kepala Divisi Manajemen Risiko sebagai anggota. Dari keseluruhan gambaran mengenai fraud sebagaimana telah
digambarkan diatas hal yang paling penting dimiliki oleh seluruh personil pegawai
bank adalah Integritas. Integritas adalah suatu konsep yang berkaitan dengan
konsistensi dalam tindakan, nilai-nilai, metode, ukuran, prinsip, ekspektasi, dan
berbagai hal yang dihasilkan. Pegawai bank yang berintegritas berarti memiliki
pribadi yang jujur dan memiliki karakter kuat. Mengapa penilaian integritas
diutamakan dimiliki oleh pegawai bank, hal ini bertujuan agar pegawai bank
mampu menolak dan menghindari untuk melakukan fraud meskipun mendapatkan
godaan dari Nasabah atau pihak eksternal lainnya. Didalam Kamus Besar Bahasa
Indonesia Integritas diartikan sebagai mutu, sifat, atau keadaan yang menunjukkan
kesatuan yang utuh sehingga memiliki potensi dan kemampuan yang memancarkan
kewibawaan dan kejujuran. Permasalahan yang muncul terjadi saat ini adalah
peranan integritas itu sendiri di dalam perilaku moral yang dapat menjawab hal-hal
yang berkaitan dengan permasalahan fraud (kecurangan). Permasalahan moral dan integritas profesional perbankan lebih
dipengaruhi oleh faktor sumber daya manusianya, mereka terjebak keinginan
mereka baik pribadi maupun kelompok untuk melakukan tindakan fraud dengan
menyalahgunakan kewenangan, kesempatan, dan sarana yang ada padanya karena
jabatan atau kedudukan. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Layanan eKYC
seperti IdentifAI yang dikembangkan oleh Nodeflux memungkinkan proses
KYC yang lebih cepat dan efisien, calon pelanggan atau nasabah hanya perlu
mengambil swa foto (selfie), lalu mengunggah foto tersebut beserta Nomor
Induk Kependudukan (NIK) melalui API atau Web Interface, data yang
diunggah tersebut kemudian akan dicocokan (matching) dengan database 1:N
ke lebih dari 190 juta data kependudukan yang dikelola oleh Dukcapil, setelah
itu akan muncul "similarity result" yang akan dijadikan sebagai acuan pada
proses verifikasi. Melalui eKYC verifikasi nasabah dilakukan secara online,
sehingga pemalsuan data akan sulit dilakukan dan pencucian uang yang
merupakan salah satu bentuk fraud dapat dicegah. p
f
p
g
7. Whistleblower
Salah satu deteksi yang dilakukan oleh Bank adalah melalui whistleblower. Melalui whistleblower, Bank memberi ruang bagi pihak internal dan pihak
eksternal, meliputi nasabah dan vendor untuk melaporkan kasus fraud kepada
manajemen, ketika mengetahui bahwa seseorang melakukan fraud. Nama
pihak
pelapor
(whistleblower)
akan
dirahasiakan
dan
tindakan
sebagai whistleblower, khususnya pihak karyawan tidak menjadi suatu catatan
buruk di konduite karyawan tersebut. 7. Whistleblower
Salah satu deteksi yang dilakukan oleh Bank adalah melalui whistleblower. Melalui whistleblower, Bank memberi ruang bagi pihak internal dan pihak
eksternal, meliputi nasabah dan vendor untuk melaporkan kasus fraud kepada
manajemen, ketika mengetahui bahwa seseorang melakukan fraud. Nama
pihak
pelapor
(whistleblower)
akan
dirahasiakan
dan
tindakan
sebagai whistleblower, khususnya pihak karyawan tidak menjadi suatu catatan
buruk di konduite karyawan tersebut. y
Penerapan anti-fraud di Bank melibatkan dan merupakan tanggung jawab
setiap individu yang terdapat di Bank yaitu Karyawan, Direksi, dan Dewan
Komisaris. Bank telah menyusun Kebijakan Anti Fraud, Kebijakan Benturan
Kepentingan, Kebijakan Know Your Employee dan Pedoman serta Tata Tertib Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Aspek perilaku individu yang mempengaruhi sumber daya
manusia dalam fraud adalah adalah faktor internal yang lebih dominan, yaitu
perilaku sebagai berikut : 1. Sifat Tamak/ Rakus Manusia, mempunyai hasrat memperkaya diri sendiri; 2. Moral yang kurang kuat, moralnya kurang kuat mudah tergoda untuk
melakukan tindak fraud. Godaan bisa datang dari berbagai pengaruh di
sekelilingnya, seperti atasan, rekan kerja, bawahan, atau pihak lain yang
memberi kesempatan; 3. Gaya hidup yang konsumtif, gaya hidup mewah di kota-kota besar mendorong
seseorang untuk berperilaku konsumptif. Perilaku konsumtif yang tidak
diimbangi dengan pendapatan yang sesuai, menciptakan peluang bagi
seseorang untuk melakukan tindak fraud; 4. Memiliki hutang yang besar, memiliki hutang atau kewajiban yang besar
tentunya akan lebih terdorong untuk melakukan segala cara agar mereka dapat
segera melunasi hutang tersebut. g
g
Secara konseptual, integritas dan nilai etika sangat jelas memberikan
pengaruh positif pada pegawai bank. Hal yang lebih penting adalah bagaimana
integritas dan nilai etika perbankan dapat diwujudkan dan ditegakkan, tentunya
harus menterjemahkannya ke dalam aturan-aturan, perilaku, serta penerapannya Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 23 Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… Rio Vernika Putra, Upaya Pencegahan Fraud Pada Bank…… 23 dalam kegiatan sehari-sehari secara konsisten oleh semua sumber daya manusia
yang tersedia. Integritas di dalam etika merupakan bentuk kejujuran dan kebenaran dari
tindakan seseorang. Integritas profesional pada diri seseorang adalah : 1. Komitmen dan loyalitas yang tercermin dalam tindakan seseorang yang
berkomitmen adalah mereka yang dapat menepati sebuah janji dan
mempertahankan janji itu sampai akhir. Faktor pemicu gagalnya seseorang
berkomitmen mulai dari keyakinan yang goyah, gaya hidup yang tidak benar,
pengaruh lingkungan, hingga ketidakmampuan mengatasi berbagai persoalan
kehidupan. Gagal dalam komitmen menujukkan lemahnya integritas diri; 2. Tanggung jawab adalah tanda dari kedewasaan pribadi. Orang yang berani
bertanggung jawab adalah mereka yang bersedia mengambil risiko,
memperbaiki keadaan, dan melakukan kewajiban dengan kemampuan terbaik 3. Dapat dipercaya, jujur dan setia. Kehidupan kita akan menjadi dipercaya,
apabila perkataan kita sejalan dengan perbuatan kita; tentunya dalam hal ini
yang kita pandang baik atau positif; 4. Konsisten berarti tetap pada pendirian. Konsiten adalah orang yang tegas pada
keputusan dan pendiriannya tidak goyah, keputusan yang diambil beradasarkan
fakta yang akurat, tujuan yang jelas dan pertimbangan yang bijak. Selalu ada
harga yang harus dibayar untuk sebuah konsistensi dimulai dari penguasaan
diri dan sikap disiplin; 5. Menguasai dan mendisiplinkan diri berarti melakukan yang seharusnya
dilakukan, bukan sekedar hal yang ingin dilakukan. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Disiplin mencerminkan
sikap pengendalian diri, suatu sikap hidup yang teratur dan seimbang; 6. Berkualitas, kualitas hidup seseorang itu sangat penting. Kualitas menentukan
kuantitas. Bila diri seseorang berkualitas maka hidupnya tidak akan
diremehkan. Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 24 baik, (4) Transparansi kepada Nasabah, (5) Tingkatkan Pengawasan Personil, (6)
Terapkan e-KYC, (7) Whistleblower. Aspek moral dan integritas harus dimiliki
oleh setiap pihak yang terlibat dalam kegiatan di bank. Selain itu, Aparat Penegak
Hukum juga haru mematuhi penerapan strategi anti-fraud bank umum yang telah
diatur dalam POJK guna Penyelamatan Keuangan Negara dan Aset Negara serta
Pemulihan Ekonomi Nasional. Bank berplat merah diharapkan tidak lalai untuk
mengikuti aturan yang ada. Selain itu diperlukan peraturan tambahan atau kebijakan
yang sesuai dengan perkembangan jaman dan teknologi agar peraturan atau
kebijakan tersebut sesuai dengan kondisi saat ini, sehingga kejahatan dalam
perbankan terutama fraud dapat diminimalisir. Jurnal Gamlath Mohottige, et.all. 2018, “The New Fraud Triangle Theory - Integrating
Ethical Values Of Employees”, International Journal of Business, Economics
and Law, Vol. 16, Issue 5 (August), p. 52-57. Vijayalakshmi P, et.all, 2021, “Impacts Of Cyber Crime On Internet Banking”,
International Journal of Engineering Technology and Management Sciences,
Issue: 2 Volume No.5 March – 2021, p. 30-34. Internet Muhammad Choirul Anwar, 2020, “ Tersangka Kasus Suap Miliaran, Eks Dirut
BTN
Ditahan
Kejagung”,
https://www.cnbcindonesia.com/market/20201010141502-17-
193389/tersangka-kasus-suap-miliaran-eks-dirut-btn-ditahan-kejagung. Diakses pada tanggal 2 Oktober 2020. Buku ACFE, 2020, Report To The Nations 2020 Global Study on Occupational Fraud
and Abuse, US, Association of Certified Fraud Examiners. C. Penutup Berdasarkan uraian hasil penelitian dan pembahasan diatas dapat
disimpulkan, bahwa Bank Milik Negara yang tersandung fraud (kecurangan)
tergolong masih tinggi jumlahnya pada setiap tahunnya dimana akibat dari fraud
telah merambah lebih luas lagi yang mengakibatkan kerugian keuangan negara. Salah satu hal yang harus diperhatikan dalam membuat strategi anti-fraud adalah
bank wajib memperhatikan : a. Kondisi lingkungan internal dan eksternal; b. Kompleksitas kegiatan usaha; c. Potensi, jenis, dan resiko fraud; Kecukupan sumber daya yang dibutuhkan e. Pembinaan karakter sumber daya sehingga memiliki nilai dan berkarakter baik;
f. Integritas dari pegawai bank sangat penting untuk mendukung banking e. Pembinaan karakter sumber daya sehingga memiliki nilai dan berkarakter baik; Pembinaan karakter sumber daya sehingga m y
gg
f. Integritas dari pegawai bank sangat penting untuk mendukung banking
prudential principle guna mewujudkan perbankan yang sehat (wellness) dan
perbankan yang patuh (comply). Adapun upaya pencegahan upaya pencegahan terdiri dari aspek teknis dan
moral serta integritas. Aspek teknis dapat melalui (1) Cek ulang data nasabah, (2)
Memberikan Reward Kepada Penemu Celah, (3) Ciptakan kontrol internal yang Jurnal Magister Hukum PERSPEKTIF, Volume 12, No. 1, April, Tahun 2021 Peraturan perundang-undangan Peraturan Bank Indonesia (PBI) No.5/8/PBI/2003 tanggal 19 Mei 2003 tentang
Penerapan Manajemen Risiko bagi Bank Umum. Peraturan OJK Nomor 39/POJK.03/2019 tentang Penerapan Strategi Anti-Fraud
Bagi Bank Umum. Surat Edaran Edaran Bank Indonesia No. 13/28/DPNP perihal Penerapan Strategi
Anti Fraud bagi Bank Umum sebagai upaya penguatan sistem pengendalian
intern bank, pengawasan aktif manajemen, struktur organisasi dan
pertanggungjawaban.
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Anmeldelse af Lars Albinus, At være tanken tro. Religion i filosofihistorisk kontekst
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RvT 70 (2020) 240-242
Anmeldelse
Lars Albinus, At være tanken tro. Religion i filosofihistorisk kontekst,
Aarhus: forlaget Klim 2019, 297 sider, kr. 299,95.
Til at varetage disciplinen religionsfilosofi er Religionsvidenskab i Aarhus så heldig
at have en af landets skarpeste, filosofiske hoveder. Lars Albinus slog med disputatsen Religion, magt og kommunikation (2010) sit navn fast som en fremragende kender
af Foucault og Habermas. Senere har han uegennyttigt kastet sig over det ikke specielt
tillokkende arbejde med at skrive lærebøger – først til det grundlæggende kursus i
filosofi og videnskabsteori (bogen Studium generale fra 2012), og nu har vi så lærebogen i religionsfilosofi, hvis primære funktion er at være “ledsager til et kompendium”
af primærtekster, som er opregnet på s. 12 f. Disse tekster spænder fra Anselm til
Agamben, men vi får ikke noget at vide om de kriterier, ud fra hvilke de er blevet
valgt.
Heldigvis giver bogens opbygning i tre hoveddele et indirekte svar: Første del
(“Religion fra Gud til menneske”) fører læseren fra antikken (Platon og Aristoteles)
over skolastikkens gudsbeviser og ‘rationalitetens Gud’ (hos Descartes, Spinoza og
Kant) frem til det 19. århundredes religionskritik. Anden del (“Religionsfilosofi efter
den sproglige vending”) har rimeligt nok Wittgenstein som den absolutte hovedskikkelse; men der bliver også plads til mere hermeneutisk orienterede tænkere som Cassirer og Ricoeur. Endelig følger den relativt korte, tredje hoveddel (“Religion og samfund”) med omtale af Foucault og Agamben, hvorefter det hele slutter med “Afrunding og resumé”.
Der er tale om en imponerende præstation. Skønt der langt fra er tale om nogen
læse-let-bog, fører Albinus læseren sikkert og suverænt igennem de mange komplekse forfatterskaber og tankegange. Både som underviser og skriftlig formidler er
han i besiddelse af en respektindgydende belæsthed og tankehorisont, som i bogen
bl.a. kommer til udtryk i en række perspektiverende og uddybende tekst-bokse, samt
(for den særligt interesserede) i slutnoterne efter hvert kapitel. Og så har jeg slet ikke
nævnt det fremragende appendiks betitlet “Spinoza og den tyske idealisme”. Umiddelbart virker det overraskende, at ballet åbnes med en længere fremstilling af Hume.
Albinus får det dog til at give rigtig god mening, for sandt er det jo, at Hume er den
første modernitetstænker, der stiller kritiske spørgsmål til sin samtids religion (åbenbaring, mirakler, etc.). 1 Som sagt: Det hele er udført med imponerende overblik, systematisk klarhed og en evne til at trække relevante og indsigtsgivende forbindelseslinjer. Men nu til nogle overvejelser (ment som åbne spørgsmål), som læsningen har
efterladt mig med.
Titlen At være tanken tro først: Den er smuk – og jeg fornemmer, at den også står
som lidt af et personligt motto for Albinus selv. 2 Alligevel kommer den i mine ører
(for?) tæt på grænsen til det patetiske. Så hvorfor ikke blot lade titlen afspejle den
disciplin, den er lærebog for, nemlig religionsfilosofi? Svaret ligger i det spændingsfyldte og (stadig) kontroversielle forhold mellem teologi og religionsvidenskab. Albinus, der søger at honorere denne forskel gennem en sondring mellem religion som
anliggende og religion som fænomen, skriver: “Selvom jeg ikke i sidste ende betragter
det som muligt at skille disse to niveauer fra hinanden, trækker nærværende bog ikke
desto mindre en streg i sandet [!] og overlader spørgsmålet om gyldighed til teologen
og den ‘systematiske’ religionsfilosofi” (s. 13). I samme ånd hedder det allerede på s.
9 om bogen, at den beskæftiger sig “ikke specielt med en refleksion over de religiøse
udsagns gyldighedsbetingelser”. Men spiller det ikke netop en vigtig rolle i kap. 10
om “Det religiøse sprog”?
Kort sagt: Jeg stiller mig kritisk-spørgende til Albinus’ måde at ‘varedeklarere’ bogen på; det er som om, han gør sig anstrengelser for at et etablere en væsentlig forskel
imellem teologi og religionsvidenskab, som han alligevel ikke rigtig selv tror på.
Uklart bliver det således helt til slut, hvor han er inde på “parløbet mellem filosofiens
og den kristne religions historie” (netop det, som er Habermas’ fokus i det nævnte
værk). Med Descartes og Kant, skriver Albinus, forlod filosofien “religionens interne
perspektiv” og erstattede det med “tankens selvforpligtende konsekvens” (s. 266), –
altså igen temaet med ‘at være tanken tro’. Og så lyder bogens afsluttende ord således: “Religion er det, der – som udsagn, praksis, liv – overskrider kløften mellem
sprog og virkelighed. Religionen er det, der finder sted i lyset af noget andet. Og dette
andet er ikke blot religionens, men også filosofiens ufravigelige skygge. Parløbet ophørte aldrig helt” (ibid.). Uklarheden er ekstra ærgerlig på dette sted, hvor enhver
vist kan høre, at der er tale om et både vigtigt og vægtigt udsagn om hvad religion
sådan i grunden ‘er’. Men hvad der sådan mere præcist ligger i det, formår jeg som
læser/anmelder ikke at udgrunde; men jeg ville rigtig gerne have haft det uddybet! 3
Også bogens undertitel (“Religion i filosofihistorisk kontekst”) har jeg grundet lidt
over. Hvorfor fastholde det filosofihistoriske perspektiv – burde det ikke være dækket
ind med kurset i Studium Generale? Ved ikke desto mindre at fastholde dette i hele
1
2
3
I sit seneste værk, Auch eine Geschichte der Philosophie (2 bd., 2019) tilkender Habermas interessant nok
Hume en lignende nøgleposition, men her med den begrundelse, at den moderne, efter-metafysiske
filosofi står over for et valg mellem to forskellige veje: enten Humes dekonstruktion af den religiøse
tradition eller Kants forsøg på en rekonstruktion af samme. Habermas slår følge med Kant.
Jf. s. 265, hvor han modstiller hhv. troen og “tankens utrættelige, asketisk selvransagende aktivitet”.
Det er nærliggende her at sammenligne med disputatsen, hvor der i ‘digressionen’ om “Teologi og
religionsvidenskab som kritisk hermeneutik” tales med større klarhed (jf. Albinus 2010, især s. 484
og 491).
Anmeldelser 241
bogens første del tvinges Albinus til en “prioritering af gudstanken” (s. 10); men er
den specielt relevant for studerende i religionsvidenskab? Hvorfor ikke i stedet sige,
at alt det med gudsbeviser og religionskritik er noget, der er sagt tilstrækkeligt om i
den forudgående filosofiundervisning? Og så kunne undertitlen måske i stedet (ganske enkelt) have lydt: ‘Filosofiske perspektiver på religion’?
I anden hoveddel undrer det mig, at Albinus bruger så meget plads på spørgsmålet om religiøse udsagns reference (Soskice og Jeffner); det bliver let ret teknisk, og jeg
tvivler på, at det vil interessere de studerende synderligt. Det ville for mig at se være
fuldt tilstrækkeligt at tage emnet op (som det også sker) i forbindelse med den glimrende fremstilling af den sene Wittgenstein. Netop hos Wittgenstein bliver det tydeligt, at der åbner sig et frugtbart perspektiv i synet på religion som ‘livsform’, som
noget vi ‘bruger’, – som en måde at ‘se’ verden på. Og i forlængelse heraf savner jeg
i bogen en inddragelse af to filosofiske retninger, som forfatteren (formentlig bevidst?) har fravalgt: pragmatismen og fænomenologien. Jeg véd ikke hvorfor; men
måske skal vi søge forklaringen i det stærke fokus, der lægges på den sproglige vending. Men man kan vel trods alt stadig tale om ‘religiøse erfaringer’ uden eo ipso at
falde bag om den sproglige vending? – Til slut blot en lille detalje: En besynderlig fejl
har indsneget sig på s. 257, hvor regula fidei stilles over for regula vitei – og det endda
to gange i træk. Det rimer ganske vist; men det hedder regula vitae! Jeg gætter på, at
det er en ikke-latinskkyndig korrekturlæser, som uafvidende er kommet til at gøre
forfatteren en bjørnetjeneste.
De ovenfor antydede kritiske spørgsmål og overvejelser rokker ikke ved min hovedopfattelse, nemlig at Lars Albinus med At være tanken tro har leveret en både indsigtsfuld og perspektivrig fremstilling og drøftelse af en række væsentlige filosoffers
beskæftigelse med temaet ‘religion’. Gid bogen må finde mange flere læsere end de
studerende, som er forpligtede på den som lærebog, for det fortjener den!
Troels Nørager, lektor emer., dr. theol.
e-mail: troelsnorager@gmail.com
242 RvT 70, 2020
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Beyond user experience and technology acceptance: Criteria to select a technology for a road safety behavioural change intervention
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Transactions on Transport Sciences
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Transactions on Transport Sciences
Peer-Reviewed Open Access Journal
Vol. 2/2021 DOI: 10.5507/tots.2021.017
journal homepage: www.tots.upol.cz DANIEL VANKOV Queensland University of Technology, Centre for Accident Research and Road Safety – Queensland, 130 Victoria Park Road,
Kelvin Grove 4059, Queensland, Australia This theoretical study proposes an informed approach to making
that choice, particularly for road safety interventions. The approach is
grounded in the Theory of Planned Behaviour. Criteria are suggested for
initial technology impact evaluation, which should help researchers and
practitioners compare different technology examples. Further criteria
are touched upon in relation to practicalities around using technologies
in interventions. The proposed framework may help future research of
technology effectiveness by saving stakeholders’ time to decide which
technology they should adopt and test. ABSTRACT: Novel technologies, such as smartphones or virtual real-
ity, are seen as attractive tools with high potential to trigger behavioral
change. Therefore, they are embraced enthusiastically by both business
and academia. Interventions using technology as tools can be expected
to further increase their popularity in the near future. A large amount of contemporary research seems to focus on examin-
ing user experience and technology acceptance. Such a focus is a natural
first step in understanding a novel technology. At the same time, much
less is known about the actual effectiveness of novel technologies in
interventions. This limited knowledge represents a potential challenge
to researchers when deciding which particular technology example to
choose for their work. KEYWORDS: Behavioural change; Novel technologies; Selection criteria;
Smartphones; Theory of Planned Behaviour 1. INTRODUCTION For example, driving performance is negatively affected by
drugs and alcohol (Siliquini et al., 2010). Driving intoxicated
increases the risk of crashes (Hingson, Heeren, Levenson,
Jamanka, & Voas, 2002). At the same time, ethical and legal
considerations limit the opportunities for such behaviour
to be researched (Brookhuis, Waard, & Samyn, 2004). Novel
technology may help open the door for such research to be
implemented in safety (Rizzo & Koenig, 2017; Steinberger
et al., 2017; Vankov & Jankovszky, InPress; Vankov et al.,
2021a). However, to the best of the author’s knowledge, no
systematic approach to select best-suited novel technology
tools to trigger behaviour change is available. Hence, their
motivation to propose such an approach. In recent years, novel technologies (e.g. smartphones, virtual
reality, health trackers, etc.) have been introduced in many
aspects of our lives. They claim to answer wants or needs,
even when people are not consciously aware of them. Despite
such claims, technologies may not be necessarily designed
from the perspective of a positive behavioral change. While
novel technologies may be quickly adopted in specific fields,
such as education (Oyelere et al., 2020), evidence may lack
about their impact, such as the case of virtual reality in road
safety (Lang et al., 2018; Vankov & Jankovszky, InPress). y (
g
J
y
)
For example, technology applications in road safety are
readily available to the general public (Lang et al., 2018; Lee,
2007; Vankov, 2020). They explore serious gaming concepts,
i.e. they are arguably designed with a primary goal differ-
ent from mere entertainment. Gameful designs or gamifica-
tion may boost motivation and commitment to using such
technologies (Diewald, Möller, Roalter, Stockinger, & Kranz,
2013; Steinberger, Schroeter, Foth, & Johnson, 2017). Several
integrated tools (feedback, challenges, social approval, and
rewards) have the potential to trigger a desired positive impact
(Diewald et al., 2013; Vankov, Schroeter, & Twisk, 2021a). How-
ever, evidence shows that the positive effects of using such
technologies are not guaranteed (Oviedo-Trespalacios, Haque,
King, & Washington, 2016; Vankov et al., 2021a; Vankov, 2020). Understanding the impact of technologies in interventions can
be further complicated by high drop-out rates, lack of informa-
tion on participants’ previous familiarity with the technology,
or lack of comparison data (Vankov, 2020). Beyond user experience and technology acceptance:
Criteria to select a technology for a road safety
behavioural change intervention DANIEL VANKOV Transactions on Transport Sciences | Vol. 2/2021 2.1 Transtheoretical model of health behavior change 2.1 Transtheoretical model of health behavior change TTM (Prochaska & Velicer, 1997) argues that each individual
is in a different stage of behavioural change concerning a spe-
cific health-related behaviour. The theory is widely used to
explain how people engage in activities to change their be-
haviour, how they progress through the changes, and what
efforts are put in place to maintain new and better behaviour. According to the model, there are six stages of change (Pre-
contemplation (Not Ready); Contemplation (Getting Ready);
Preparation (Ready); Action; Maintenance and Termination)
(Prochaska & Velicer, 1997), with the first five being measur-
able. TTM suggests strategies based on identified individual
readiness to change, which can help a person move from
one stage to the next. The opposite process (relapse) is also
possible at any stage. However, choosing a novel technology for a road safety
intervention may often be an early-stage effort. At early stage,
it is uncommon to have detailed information about the po-
tential intervention participants that informs at what stage
of change they are. Instead, the intervention itself might be
used to collect such information. Thus, the initial interven-
tion is more likely not to be stage-tailored. If tailored, preliminary tailoring to the needs of a specific
group of participants carries the risk of the chosen target
stage not being identified correctly. Such a shortcoming may
be the result of the respective road safety behavioural process
potential oversimplification. Furthermore, in road safety,
problematic habits in many cases may be semi-automated
and, thus, might not be accounted for by the model or the
chosen technology it informs. TTM is applied in various health-related interventions,
such as stress management (Prochaska et al., 2012), reduc-
tion of smoking (Prochaska, DiClemente, Velicer, & Rossi,
1993), improving energy balance (Van Duyn et al., 1998;
Velicer et al., 2013) or obesity reduction (Mauriello et al.,
2010). In many cases, TTM is used to assess interventions’
impact on more than one dimension (e.g. technology, young
people and risky behaviour). For example, Prochaska et al. (2012) used a telephone and an online program coaching
to improve well-being through improved life evaluation,
healthy behaviour, and emotional and physical health. Ave-
yard et al. (1999) used three TTM-based computer sessions
and three class lessons over a year to reduce smoking in
young people and their peers. Velicer et al. (2013) imple-
mented a computer-based intervention to prevent substance
abuse. 2.2 Social Cognitive Theory SCT (Bandura, 1986) addresses some TTM limitations. First,
the theory looks at the behavioral process as a whole and
not as divided into stages. Thus, it can be used to inform
interventions with little initial information about its partici-
pants. Second, the model goes beyond the limitation of not
accounting for interpersonal factors, which are accounted
for by its “environment” construct. y
SCT (Bandura, 1986) suggests that humans function due
to interactions between their environment, personality and
behaviour. The theory argues that personality is developed
through observational learning and social experience. The
environment constitutes the external influences on one’s
behaviour, while the personality is one’s motivational factor
to perform the behaviour. The model’s personality dimen-
sion includes key constructs which influence performing the
desired behaviour: 1) self-efficacy or how much an individual
believes they can achieve specific goals, outcome expecta-
tions or the individual’s expectations, in case they perform
a behaviour; 2) self-control or how much an individual can
autonomously regulate their intentions and behaviours,
reinforcements or internal or external responses to an indi-
vidual’s behaviour, affecting their likelihood to continue it;
and 3) observational learning or the ability of an individual
to reproduce a behaviour after observing it in others. SCT
considers both self-reflection, or whether individuals can
critically analyse their behaviour, and potential influences
of personality characteristics within these constructs. Thus,
when evaluating potential technology candidates, a relevant
question would be “Does the technology improve the user’s
ability to understand their behaviour?” or “Does the tech-
nology change the user’s expectations when performing the
behaviour?” p
(
gg
)
Like most models, TTM has attracted criticism (Littell
& Girvin, 2002; West, 2005). Littell and Girvin (2002) see
the biggest problem in oversimplifying complex behavioural
change processes into stages. Other researchers found in-
stability of the stages themselves (De Nooijer, Van Assema,
De Vet, & Brug, 2005; Hughes, Keely, Fagerstrom, & Callas,
2005), challenging the model presumption that people make
coherent and stable plans. The model also neglects that many
health problems arise from semi-automated unhealthy hab-
its, which are not easy to change (West, 2005). For example,
Velicer et al. (2013) reported limited results in directly ad-
dressing smoking and alcohol. The authors suggested that
reactivity and defensiveness towards behavioural change
may have been due to the behaviours’ addictive nature. So,
the model can propose an incorrect intervention strategy for
some behaviours (West, 2005). 2.1 Transtheoretical model of health behavior change Thus, TTM is identified as a helpful model in multidis-
ciplinary research, including human-computer interaction
(Aveyard et al., 1999; Di Noia, Contento, & Prochaska, 2008;
Gold et al., 2016) and persuasive technologies that change
users’ behaviours and attitudes through social influence and
persuasion (Fogg, 2009). Finally, TTM focuses on the individual and not on dynamic
interpersonal interactions, which are common in road safety. Accounting for interpersonal relations is better to be consid-
ered so that normative influences for achieving a positive
change can be assessed. Thus, TTM might not be the best
initial choice to assess the suitability of novel technology
tools to trigger behaviour change. Transactions on Transport Sciences | Vol. 2/2021 2. UNDERSTANDING BEHAVIOUR CHANGE Research into novel technologies starts with examining user
experience and technology acceptance (Vaezipour, 2018;
Vankov & Jankovszky, InPress). After this knowledge is es-
tablished, the critical question “What is the potential of such
technology to impact an individual’s behaviour?” remains. To
answer this question, an appropriate theoretical grounding
can provide a sound basis for an in-depth evaluation, which,
in turn, may provide insights on how the desired behaviour-
al change could potentially be motivated (Kohler, Grimley,
& Reynolds, 1999). Many theories are applied to explore behaviour in scientific
interventions in various fields, such as health, human-com-
puter interaction or road safety. A systematic review of 256
articles (8,680 initially retrieved) identified 82 theories in the
social and behavioural sciences literature (Davis, Campbell,
Hildon, Hobbs, & Michie, 2015). Three of them accounted for
57% of the reviewed articles: Transtheoretical model of health Regardless of limitations, technology can add value to road
safety research. Such research often tries to influence the
behavior of traffic participants to prevent risky situations. Transactions on Transport Sciences | Vol. 2/2021 53 behavior change (TTM) (Prochaska & Velicer, 1997) (33%),
Theory of Planned Behaviour (TPB) (Ajzen, 1991) (13%), and
Social Cognitive Theory (SCT) (Bandura, 1986) (11%). Any of
those theories can inform the selection of a technology that
has the potential to influence a targeted behaviour positively
when utilised as an intervention tool. the user understand at which stage of change they are?” or
“Does the technology help the user change their behaviour to
move from one stage to another?” Dividing a participant pool into subgroups, based on their
stage, may further allow understanding how effective a re-
spective intervention is in the case of each different stage,
i.e. by assessing whether and how far the participants from
the respective stage of change progressed as a result of the
intervention. As a result, the model could provide valuable
information for what type of participants such interventions
could deliver maximum benefit. 2.2 Social Cognitive Theory Regardless of its limitations, TTM could be considered
a guiding model to choose best-suited novel technology tools
to trigger behaviour change, i.e. tools designed to push par-
ticipants from one stage to another regarding their risky
behaviour. As mentioned earlier, TTM can help measure five
different stages. The advantage of TTM is that it aids under-
standing at which stage of change the study participants are,
particularly when dealing with groups that are not homog-
enous. Thus, relevant questions when evaluating potential
technology candidates would be “Does the technology help SCT has a long track record of being applied in various
fields. The theory informed studies concerned with health Transactions on Transport Sciences | Vol. 2/2021 54 act a behaviour of interest (Mackenzie, 2016). Unconscious
influences on behaviour (Sheeran, Gollwitzer, & Bargh, 2013)
are also pointed out as a major limitation, as TPB exclusively
focuses on rational reasoning. Other researchers underline
as a limitation the TPB static explanatory nature (Sniehotta,
Presseau, & Araújo-Soares, 2014). Sniehotta et al. (2014)
also suggest that the main problem of TPB is in the valid-
ity of its predictions as the sequence of influences it ex-
plores conflicts with some available evidence. For example,
as a consequence of intervention, shifts in behaviour are
not always moderated directly through the TPB constructs
(Webb & Sheeran, 2006). Sometimes, when a habit drives the
behaviour, reverse causation is possible (Webb & Sheeran,
2006). In such cases, intention has little influence on be-
haviour, and past behaviour is a much stronger predictor
of intention and future behaviour (Fishbein & Ajzen, 2010;
Sommer, 2011). risks prevention (Miller, Shoda, & Hurley, 1996; Schwarzer
& Renner, 2000; Wallace, Buckworth, Kirby, & Sherman,
2000), human-computer interaction (Compeau & Higgins,
1995; Ifinedo, 2016), young people (Ifinedo, 2016; Wallace et
al., 2000) and their peers (Compeau & Higgins, 1995; Ifinedo,
2016; Rana & Dwivedi, 2015; Wallace et al., 2000). While recognising its relative utility, some researchers see
SCT as a collection of logical statements that are difficult to
test empirically (Smedslund, 1978). Other researchers see its
constructs as based on variables that are not well defined and
cannot be observed and assessed (Lee, 1989). For example,
the instruments to measure self-efficacy may not be carefully
developed and validated (Frei, Svarin, Steurer-Stey, & Puhan,
2009). In addition, Mackenzie (2016) questioned SCT’s ability
to account for motivation at the moment of executing the
behaviour. 2.3 Theory of Planned Behaviour 2.3 Theory of Planned Behaviour According to TPB (Ajzen, 1991), future behaviour is best pre-
dicted by intention to behave as its immediate antecedent. Intention, in turn, is predicted by three interrelated factors:
1) attitude or whether a behaviour is seen as favourable or
unfavourable; 2) subjective norm or whether significant oth-
ers are seen as approving or disapproving the behaviour;
and 3) perceived behavioural control (PBC) or whether the
behaviour is seen as easy or challenging to perform (Ajzen,
1991). PBC is also seen as directly predicting behaviour. The TPB components have been used to explain various
risk-related behaviours in the health domain, e.g. safer sex,
uptake of vitamin C or cycle helmet use (Rutter & Quine,
2002). Rutter and Quine (2002) overview of evidence suggests
that, on average, around 40% of the variance in both health
behaviour and intention can be explained through TPB. Exploring all 82 theories or even using the three major
ones to select technology as an intervention tool in road
safety is beyond the scope of the current article. Thus, based
on the above discussion, only an approach building on TPB
(Ajzen, 1991), which accounted for 13% of the articles, second
in the ranking (Davis et al., 2015), is discussed in turn. TPB addresses some of the suitability issues identified in
TTM and SCT. TPB is not stage-tailored and accounts for in-
terpersonal relations through its subjective norm, addressing
the discussed-above TTM limitations of being stage-tailored
and focusing on the individual. Despite not being stage-tai-
lored, TPB allows for tailoring strategies to encourage specific
behaviour, for example, through interventions, as in Quine,
Rutter, and Arnold (2001). There are also validated question-
naires in the TPB toolset, as in Lennon, Oviedo-Trespalacios,
and Matthews (2017), Chen et al. (2016), Haque et al. (2012),
Elliott and Thomson (2010) and Quine et al. (2001), address-
ing the discussed SCT limitation of lack of carefully developed
and validated instruments. 2.2 Social Cognitive Theory The SCT could be considered a guiding model to choose
best-suited novel technology tools to trigger behaviour
change. The model can inform interventions’ evaluation
design and can provide insights into the participants’ self-
efficacy (e.g. how to support personal confidence in achiev-
ing behaviour change results or what information to be pro-
vided to increase self-reflection), and normative influences
(e.g. how to shape the intervention environment to encourage
safe behaviour on the road). However, the lack of carefully
developed and validated measurement instruments is a no-
table limitation. Developing and validating questionnaires
is more likely to happen after a technology is already chosen
as a tool than before that. In addition, the technology could
influence participants when they perform risky behaviour. The SCT’s questioned ability to account for motivation at
the moment of executing the behaviour would challenge its
suitability to inform the choice of such technology in the first
place. As a result, similar to TTM, SCT might not be the best
initial choice to assess the suitability of novel technology
tools to trigger behaviour change. Some of those limitations can be addressed through re-
search design. For example, the static nature limitation could
be addressed by a longitudinal design of the studies, explor-
ing shifts in the TPB constructs over time, as in Quine et al. (2001) and Stead et al. (2005). Furthermore, using a technol-
ogy lends itself to a longitudinal design, making the limita-
tion addressable if the choice of technology accounts for it
in the first place. The assumption that people have the needed resources and
skills to enact a behaviour could be addressed by providing
research participants with additional resources and skills
that could enable them to perform the behaviour of interest. Such resources and skills can be delivered through novel
technologies (Steinberger et al., 2017), which support using
the TPB for assessing the suitability of novel technology tools
to trigger behaviour change. Transactions on Transport Sciences | Vol. 2/2021 2.4 Selecting a theory for a road safety intervention All three theories discussed above have a history of being
used in the domain of road safety. For example, TTM (Prochas-
ka & Velicer, 1997) was used to assess interventions for drink
drivers (Polacsek et al., 2001), recidivist drink drivers (Free-
man et al., 2005) and occupational road safety (Banks, 2008). SCT-based (Bandura, 1986) research investigated mature driv-
ers’ attitudes towards speeding and traffic rules violations
Tranter and Warn (2008), thrill-seeking, attitudes towards
speeding and driving violations (Yıldırım-Yenier, Vingilis,
Wiesenthal, Mann, & Seeley, 2016) and young people’s risky
driving behaviour (Scott-Parker, 2012). TPB (Ajzen, 1991)
informed investigations into speeding (Elliott & Thomson,
2010; Vankov, Schroeter, & Twisk, 2021b; Warner & Åberg,
2008), driving under the influence (Chan, Wu, & Hung, 2010;
Potard, Kubiszewski, Camus, Courtois, & Gaymard, 2018;
Vankov & Schroeter, 2021) and distraction (Bazargan-Hejazi
et al., 2017; Chen et al., 2016). 3. TPB AND SELECTING A TECHNOLOGY
INTERVENTION TOOL IN ROAD SAFETY Ajzen (2006) suggests interventions be designed to influence
one or more behavioural predictors to change behaviour. Un-
fortunately, Ajzen (2006) does not specify what interventions
(e.g. media campaigns, face-to-face, etc.) may trigger the
desired behavioural change. Fife–Schaw, Sheeran, and Nor-
man (2007) agree that TPB is silent in guiding appropriate
strategies to influence its basic constructs. Thus, it is left to Similar to TTM and SCT, TPB also suffers from limitations. For example, a criticised TPB assumption is that the theory
sees the person as having all the resources and skills to en- Transactions on Transport Sciences | Vol. 2/2021 55 the researchers to establish their criteria while consider-
ing the specific needs of their work. To successfully achieve
that, they may choose to extend the theory with additional
theoretical constructs to improve their model’s predictive
validity (Elliott & Thomson, 2010; Scott-Parker, 2012; Vankov
& Schroeter, 2021; Vankov et al., 2021b). the researchers to establish their criteria while consider-
ing the specific needs of their work. To successfully achieve
that, they may choose to extend the theory with additional
theoretical constructs to improve their model’s predictive
validity (Elliott & Thomson, 2010; Scott-Parker, 2012; Vankov
& Schroeter, 2021; Vankov et al., 2021b). at all) to 7 (very much so) or 1 (definitely not) to 7 (definitely). Adding the points will determine an overall score, allowing
the technologies to be ranked. At the next assessment level,
whether binary or Likert, multiple experts can assess the
technologies. To best account for potential professional bias,
we believe that experts with diverse backgrounds should
be tasked with the assessment. For example, such expects
may come from academia, regulatory bodies, technology
companies, road safety not-for-profit activist groups, and
representatives from the potential target group. Then, the
individual scores of those experts can be averaged to deter-
mine the final score for comparison. Regardless of the number of additional constructs, which
can be used to extend TPB, an intervention cannot be ex-
pected to change all of them. For example, no intervention
can change demographic variables. Other personality charac-
teristics, such as impulsivity and sensitivity, are not seen as
potentially changeable in the short term, either (Scott-Parker,
2012). They are perceived as relatively stable. As a result,
researchers should aim to define a viable focus for their inter-
ventions to ensure potential success. 3. TPB AND SELECTING A TECHNOLOGY
INTERVENTION TOOL IN ROAD SAFETY Regardless that Ajzen
(2006) does not provide guidance on what type of intervention
should be best to use, he gives some guidance to research-
ers about their potential interventions’ focus. Ajzen (2006)
suggests that interventions should focus on salient beliefs,
such as the TPB attitude, subjective norm and PBC, as they
are readily accessible and might be influenced. i
p
Above, we provide a general guideline on approaching
the selection of technology for a particular intervention. Nevertheless, a researcher might choose a different theory or
different additional predictors depending on their needs (see
Understanding behaviour change). For example, suppose
there is detailed information about the potential partici-
pants, which would allow their stage of behavioural change
to be correctly identified. In that case, those participants can
be divided into subgroups, and TTM (Prochaska & Velicer,
1997) can be used to identify novel technologies for each
subgroup. Furthermore, if the intervention methodology
is established before choosing an appropriate technology,
new questionnaires could validate additional constructs. As a result, new SCT-based criteria can be developed. Alter-
natively, the selected constructs’ potential to mediate the
desired changes might be considered when assessing the
technologies as per the identified TPB selection criteria, as
discussed in turn. As an example for the current article, we will further build
on Ajzen’s (2006) suggestion by adding three salient beliefs on
top of the TPB attitude, subjective norm and PBC. Those three
additional constructs have a history of being used to extend
the theory beyond its original constructs (Elliott & Thomson,
2010; Vankov & Schroeter, 2021; Vankov et al., 2021b). They
are 1) peers’ norm, i.e. whether friends or peers are promoting
a right or wrong perception of behaviour (Conner & Sparks,
2005); 2) moral norm, i.e. whether a person sees engaging in
a behaviour as appropriate or inappropriate (Conner & Sparks,
2005), and 3) perceived risk, i.e. whether a person considers
the possibility to be involved in a crash or to be caught by the
police while performing specific behaviour (Ward et al., 2017). Thus, in our example, we consider six potentially changeable
constructs (attitude, subjective norm, PBC, moral norm, per-
ceived risk and peers’ norm) to be used to guide the selection
of technology for an intervention. For our TPB example, the literature agrees that PBC is
a weaker predictor of behaviour than intention (Armitage
& Conner, 2001; Fife‐Schaw et al., 2007). 3. TPB AND SELECTING A TECHNOLOGY
INTERVENTION TOOL IN ROAD SAFETY At the same time,
the evidence around predicting intention is mixed. Subjective
norm is generally seen as having lower predictive power in
TPB than attitude and PBC (Armitage & Conner, 2001; Fife‐
Schaw et al., 2007). According to Fife‐Schaw et al. (2007), at-
titude typically has the strongest predictive value. However,
evidence about constructs’ predictive power should better be
sought case by case for each behaviour of interest. As a re-
sult, researchers may wish to assign different weights, i.e. a higher number of points, to the constructs they consider
of higher importance. To establish selection criteria, we propose using the con-
structs’ definitions (see Table 1). Those selection criteria can
be formulated as binary questions, directly pointing at the re-
spective construct’s definition. A positive (yes) answer could
carry 1 point. A negative (no) answer should not bring points. Thus, the number of positive responses will determine the
relevant technology ranking. Alternatively, a Likert scale can
be employed if the compared technologies are too similar. For
example, the scale can have seven points, ranging from 1 (not g
p
To show how the above theoretical considerations can
be put into practice, we applied the guidelines in selecting
a smartphone app as a promising road safety intervention tool
in the case of speeding as a targeted behaviour. A review of Selection
criteria (SC) N
Construct
Definition
Question
SC1
Attitude
How the user sees the behaviour, favourable or
unfavourable. Does the technology help the user better see whether
their behaviour is favourable or unfavourable? SC2
Subjective
norm
Whether the user’s significant referents would approve or
disapprove their engagement in a particular behaviour. Does the technology provide information on how
the user’s important referents see their behaviour? SC3
PBC
How easy or challenging the user perceives performing
the behaviour. Does the technology improve the user’s ability to
perform the behaviour? SC4
Moral norm
Whether the user perceives the behaviour as
appropriate or inappropriate. Does the technology help the user understand
the morality of their behaviour? SC5
Peers’ norm
Whether the user’s peers are seen as disapproving or
approving of the respective participant engaging in
the behaviour. Does the technology provide information on how
the user’s peers perform the behaviour? SC6
Perceived
risk
Whether the driver perceives a risk of being involved in
a crash or being caught by the police while performing
a specific behaviour. 3. TPB AND SELECTING A TECHNOLOGY
INTERVENTION TOOL IN ROAD SAFETY Offers leaderboard. The leaderboard shows to the
driver how their peers are doing (SC5). Comparing
with their peers can help the driver assign a moral
value to their behaviour (SC4). 4. Free for users. 5. No geographic restriction. 6. Has a web interface. 7. Does not need a dongle. 1. Has problems with synchronising
with the GPS signal when on
autostart. 2. Drivers with short trips may in
general score lower than drivers
with long ones. 3. Does not have all car
manufacturers and models. SC1
SC2
SC3
SC4
SC5
SC6
Yes
No
Yes
Yes
Yes
No
1
0
2
1
1
0
Total points: 5
AAMI Safe
Driver
1. Monitors for exact speed limits, not the general
ones. The speed limits feedback can help the
driver understand whether it is favorable or
unfavorable (SC1). 2. Provides detailed after-trip feedback on a Google
map. This detailed information can potentially
help the driver understand the implications of any
changes in their behaviour, thus enabling them to
improve it (SC3). 3. Good mix of gamified elements (scores, badges). Receiving scores and badges can represent a higher
morality of the behaviour (SC4). 4. Runs in the background. 5. Does not need a dongle. 6. Free for users. 1. Cannot provide real-time
feedback. 2. Fails to record and analyse long
journeys. 3. Has problems with synchronising
with the GPS signal when on
autostart. 4. Very wide thresholds are set for
recording an offence. 5. Designed to sell an insurance
product. 6. Has problems with calculating the
overall score. SC1
SC2
SC3
SC4
SC5
SC6
Yes
No
Yes
Yes
No
No
1
0
2
1
0
0
Total points: 4
Hellas
Direct
1. Provides detailed after-trip feedback on a Google
map. This detailed information can potentially
help the driver understand the implications of any
changes in their behaviour, thus enabling them to
improve it (SC3). 2. Uses all common gamification elements (scores,
leaderboards badges etc ) Receiving scores and
1. Available only in selected
jurisdictions, i.e. not being tested in
Australia. 2. Always attached to insurance
products. 3. Looks like over-gamified – may
benefit from a “lite” version
SC1
SC2
SC3
SC4
SC5
SC6
No
No
Yes
Yes
Yes
No
0
0
2
1
1
0
Total points: 4 4. Free for users. 5. No geographic restriction. 6. Has a web interface. 7. Does not need a dongle. AAMI Safe
Driver
1. 3. TPB AND SELECTING A TECHNOLOGY
INTERVENTION TOOL IN ROAD SAFETY Does the technology provide information on the
possibility the user to be involved in a crash or to be
caught by the police while performing the behaviour? Table 1. Technology selection criteria derived from an extended TPB framework. Transactions on Transport Sciences | Vol. 2/2021 56 TPB studies focused on speeding indicates that PBC, and not
attitude, is the strongest predictor of intention (Elliott and
Thomson, 2010; Stead et al., 2005; Warner & Åberg, 2008). While Stead et al. (2005) and Elliott and Thomson (2010) pro-
vide support for attitude having the second strongest value
and confirm norms as having the lowest contribution, Warner
and Åberg (2008) found the reverse. Given that typically PBC
is the strongest predictor of intention in the case of speed-
ing, an intervention may focus on using smartphone apps
expected to influence PBC. Thus, a positive answer to the
PBC-related question should be weighted higher when using
the questions as criteria for selecting the most promising
technology example. influence speeding. In November 2017, we browsed the Goog-
le Play and iTunes app stores to identify such smartphone
software. The used terms were “road app”, “smart driving”,
“safe driving”, and “OBD game”. Sixty-six apps from the search
results in Google Play and 20 from iTunes were selected for
detailed investigation. g
The online reviews left by users in the smartphone safe-
driving apps’ marketing profiles were used to screen out
smartphone software that was unlikely to be a suitable in-
tervention tool. Thus, the number of apps to be investigated
in full detail was narrowed down to six. In Table 2, those
apps were scored using the established theoretical selection
criteria (see above). Double points were assigned to a positive
answer on the PBC-related question because PBC is typically
the strongest predictor of speeding. g
The next step would be to find out what smartphone soft-
ware is available that might serve as an intervention tool to Name
Pros
Cons
Scoring
Flo - driving
insights
1. Can provide live feedback to the driver or can
run invisibly in the background. The life feedback
can help the driver understand their real-time
behaviour and potentially inform conclusions
whether it is favorable or unfavorable (SC1). 2. Provides detailed after-trip feedback on a Google
map. This detailed information can potentially
help the driver understand the implications of any
changes in their behaviour, thus enabling them to
improve it (SC3). 3. 3. TPB AND SELECTING A TECHNOLOGY
INTERVENTION TOOL IN ROAD SAFETY Monitors for exact speed limits, not the general
ones. The speed limits feedback can help the
driver understand whether it is favorable or
unfavorable (SC1). 2. Provides detailed after-trip feedback on a Google
map. This detailed information can potentially
help the driver understand the implications of any
changes in their behaviour, thus enabling them to
improve it (SC3). 3. Good mix of gamified elements (scores, badges). Receiving scores and badges can represent a higher
morality of the behaviour (SC4). 4. Runs in the background. 5. Does not need a dongle. 6. Free for users. 1. Cannot provide real-time
feedback. 2. Fails to record and analyse long
journeys. 3. Has problems with synchronising
with the GPS signal when on
autostart. 4. Very wide thresholds are set for
recording an offence. 5. Designed to sell an insurance
product. 6. Has problems with calculating the
overall score. SC1
SC2
SC3
SC4
SC5
SC6
Yes
No
Yes
Yes
No
No
1
0
2
1
0
0
Total points: 4
Hellas
Direct
1. Provides detailed after-trip feedback on a Google
map. This detailed information can potentially
help the driver understand the implications of any
changes in their behaviour, thus enabling them to
improve it (SC3). 2. Uses all common gamification elements (scores,
leaderboards, badges, etc.). Receiving scores and
badges can represent a higher morality of the
behaviour (SC4). 3. Offers a leaderboard. The leaderboard shows
to the driver how their peers are doing (SC5). Comparing with their peers can help the driver
assign a moral value to their behaviour (SC4). 4. Does not need a dongle. 5. Runs in the background. 6. Free for users. 1. Available only in selected
jurisdictions, i.e. not being tested in
Australia. 2. Always attached to insurance
products. 3. Looks like over-gamified – may
benefit from a “lite” version. 4. Difficult to advise when the user is
not the driver. 5. Does not have a user-start option. SC1
SC2
SC3
SC4
SC5
SC6
No
No
Yes
Yes
Yes
No
0
0
2
1
1
0
Total points: 4 4. Runs in the background. 5. Does not need a dongle. 1. Available only in selected
jurisdictions, i.e. not being tested in
Australia. 2. Always attached to insurance
products. 3. Looks like over-gamified – may
benefit from a “lite” version. 4. Difficult to advise when the user is
not the driver. 5. Does not have a user-start option. 3. TPB AND SELECTING A TECHNOLOGY
INTERVENTION TOOL IN ROAD SAFETY SC1
SC2
SC3
SC4
SC5
SC6
No
No
Yes
Yes
Yes
No
0
0
2
1
1
0
Total points: 4 Transactions on Transport Sciences | Vol. 2/2021 57 SafeDrive
1. Blocks calls and texts. Thus, it helps the driver
perform the behaviour (SC3). 2. Free and not geographically restricted. 3. Tries to connect gamification (earning points)
with the real world (receiving rewards). Received
points can represent a level of morality of the
behaviour (SC4). 4. Works on auto-start. 1. Does not monitor other data than
mobile phone usage data. 2. No real rewards besides discounts. 3. Has problems with setting user
info. 4. Has problems with synchronising
with the GPS signal. SC1
SC2
SC3
SC4
SC5
SC6
No
No
Yes
Yes
No
No
0
0
2
1
0
0
Total points: 3
Automatic
1. Uses accurate vehicle data. The accurate vehicle
data can help the driver understand whether it is
favorable or unfavorable (SC1). 2. Provides detailed feedback. This detailed
information can potentially help the driver
understand the implications of any changes in their
behaviour, thus enabling them to improve it (SC3). 3. Assist with crash alert (SC6). 4. Diagnoses the car. 1. Costs 99.99 AUD. 2. Requires a dongle. 3. Restricted to the US. 4. Does not support all car models. SC1
SC2
SC3
SC4
SC5
SC6
Yes
No
Yes
No
No
Yes
1
0
2
0
0
1
Total points: 4
Rookie
Dongle
1. Uses accurate vehicle data. The accurate vehicle
data can help the driver understand whether it is
favorable or unfavorable (SC1). 2. Notifies parents or guardians about reckless
driving. Thus, it may trigger information on how
driver’s important referents see their behaviour
(SC2). 3. Provides detailed feedback. This detailed
information can potentially help the driver
understand the implications of any changes in their
behaviour, thus enabling them to improve it (SC3). 4. Does not require a smartphone as it has a built-
in GPS and mobile connection. 1. Costs 338.99 EUR with a one-year
subscription. 2. May discourage adoption because
of notification to third parties. SC1
SC2
SC3
SC4
SC5
SC6
Yes
Yes
Yes
No
No
No
1
1
2
0
0
0
Total points: 4
Table 2. Smartphone safe-driving apps (yes=1, no=0, double points for SC3). Table 2. Smartphone safe-driving apps (yes=1, no=0, double points for SC3). Table 2. Smartphone safe-driving apps (yes=1, no=0, double points for SC3). 3. TPB AND SELECTING A TECHNOLOGY
INTERVENTION TOOL IN ROAD SAFETY been a good candidate for a road safety intervention. Nev-
ertheless, those criteria might not be equally applicable to
all technologies or risky behaviours. For example, simula-
tor sickness is a known issue in simulator studies (De Win-
ter, van Leeuwen, & Happee, 2012). This problem seems not
applicable for smartphones but persistent in virtual reality
(Vankov & Jankovszky, InPress). As a result, simulator sick-
ness should be investigated when assessing the suitability
of both driving simulators and virtual reality applications. Thus, we recommend when the theoretically-informed suit-
ability of novel technology tools to trigger behaviour change
is being assessed by experts, as discussed above, those same
experts to be asked to provide opinions on potential practical
criteria, too. Out of the six apps, “Flo – driving insights” scored the
highest, 5. Thus, it would be the initial best candidate for
driving tests as established by the adopted theory-based cri-
teria. However, theory can provide a well-informed direction,
which may be challenging to implement in practical terms. To overcome known difficulties, a focus group, representing
a convenience sample of academia (3 people), applied re-
search (1 person) and project leaders (6 people) in the domain
of awareness-raising road safety interventions for young driv-
ers, suggested practical criteria which may boost safe-driving
apps’ adoption if met (Vankov, 2020) (see Table 3). Table 3. Synthesised practical criteria for smartphone safe-driving
apps (Vankov, 2020). Criteria
Description
Low-cost
To boost adoption, it would be best if the app is free
for users and does not require hardware, such as
a dongle, in addition to the smartphone itself. Safety
The app should provide live feedback to the driver
and run invisibly in the backgrwwound, thus, not
causing additional distraction. It also shall have
a self-starting capability. Availability
The app shall not be geographically restricted. Information
The app shall provide various types of information. It shall have a web interface. It shall provide detailed
after-trip feedback on a Google map. It should also
feature user groups (i.e. proprietary leaderboards)
with the achievements being possible to share on
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factors which influence individuals’ drink walking intentions. Safety Science, 50(3), 378-384. doi: https://doi.org/10.1016/j. ssci.2011.09.017 Brookhuis, K., Waard, D., & Samyn, N. (2004). Effects of MDMA
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440-445. doi: 10.1007/s00213-003-1714-5 Hingson, R., Heeren, T., Levenson, S., Jamanka, A., & Voas, R. (2002). 4. CONCLUSION Available technology provides opportunities to help address
social issues such as risky road behaviours. Those opportu-
nities are vastly explored by both academia and business. However, it is not uncommon to have multiple technologies
claiming to serve a similar purpose. Thus, researchers may
find it challenging to select one technology over another. Fur-
thermore, technology is often initially researched with regards
to user experience and technology acceptance. Much less is
known about their potential to influence behaviour. The current article offers researchers a helpful model to
choose a technology that best meets their needs. The model
is grounded in TPB, thus providing a robust theoretical un-
derpinning. At the same time, we show the model’s flexibility,
which can be extended depending on the specific research
needs. Then, we presented how selection criteria for choosing
a technology are developed based on six constructs (attitude,
subjective norm, PBC, moral norm, perceived risk and peers’ Table 3. Synthesised practical criteria for smartphone safe-driving
apps (Vankov, 2020). As presented in Table 2, “Flo – driving insights” complies
with all four criteria listed in Table 3. Thus, it might have Transactions on Transport Sciences | Vol. 2/2021 58 the 5th International Conference on Automotive User Interfaces
and Interactive Vehicular Applications. the 5th International Conference on Automotive User Interfaces
and Interactive Vehicular Applications. norm). Finally, we complemented the theory-based criteria
with practical considerations to inform a final choice. Re-
searchers might find the proposed approach insightful in their
future work even if they choose to apply a different theory. As a result, the provided methodologically sound yet easy to
implement guidance could potentially increase the quality of
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Human capital of the Karelian Arctic in the implementation of the special economic regime of the region
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Human capital of the Karelian Arctic in the
implementation of the special economic regime
of the region 1The institute of Economics of the Karelian Scientific Centre of the Russian Academy of Sciences,
RAS, 50 A. Nevsky Ave., 185030, Petrozavodsk, Republic of Karelia, Russia Abstract. This article presents the results of a field study of the state and
development of the human capital in the Karelian Arctic as a factor in
sustainable development of the region during the transition to a new
economic and legal regime. The focus of the scientific research is the
assessment by the citizens of the level of their well-being and the ability to
meet various family needs, personal income planning possibilities,
correspondence of the current place of work to the training received in an
educational institution. Brief conclusions are made on the indicated aspects
of the development of the human capital in the Karelian Arctic region. Data were obtained and an analysis of the situation was made both for the
Karelian Arctic as a whole, and for individual municipal districts included
in this region. Further research activities to deepen scientific knowledge
about the state and trends in the development of the human capital in
Arctic Karelia and the Arctic zone of Russia as a whole have been
identified. The issues under study are one of the key ones in determining
the parameters of the created special economic and legal regime, which
applies to Arctic Karelia. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 217, 07028 (2020)
ERSME-2020 E3S Web of Conferences 217, 07028 (2020)
ERSME-2020 https://doi.org/10.1051/e3sconf/202021707028 1 Introduction The development and adoption of strategic documents for the development of the Arctic
zone of the Russian Federation (hereinafter the AZRF), the updating of the management
tools for regional development in the context of the implementation of a new special
economic and legal regime and the entry of new territories into the AZRF require updating
and improving knowledge about the socio-economic processes in the Russian Arctic. Thus,
in particular, in the Strategy for the Development of the AZRF until 2035, the
implementation of the main tasks in the field of economic development of the Arctic zone
is assumed through the implementation of measures that include:
introduction of a special economic regime in the Arctic zone, contributing to the
transition to a circular economy, private investment in geological exploration, creation of
new and modernization of existing industrial productions, development of science-intensive
and high-tech industries, etc. ΎCorresponding author: kov8vol@gmail.com ΎCorresponding author: kov8vol@gmail.com E3S Web of Conferences 217, 07028 (2020)
ERSME-2020 https://doi.org/10.1051/e3sconf/202021707028
adjusting the system of basic professional educational programs and admission targets
for training at the expense of budgetary allocations from the federal budget, budgets of the
constituent entities of the Russian Federation, local budgets to educational organizations
located in the Arctic zone, in accordance with the demand forecast for qualified and highly
qualified personnel;
systematic provision of state support measures to the economically active population of
Russia ready to relocate (resettle) to the Arctic zone in order to carry out labor activities [1].
systematic provision of state support measures to the economically active population of
Russia ready to relocate (resettle) to the Arctic zone in order to carry out labor activities [1]. The full implementation of these measures is possible only on the basis of the
development of new management mechanisms that are most effective in using the existing
resources for the development of the Arctic regions, taking into account the existing
economic, legal, political, socio-demographic, technological and other constraints [2]. Nevertheless, the human capital of the territories is one of the most important development
resources. The relevance of the study of the Karelian Arctic is dictated primarily by the fact
that this region has become part of the AZRF relatively recently [3,4], as well as by the
novelty of the format of the proposed development in the context of global projects for the
development of the Russian Arctic. In addition, the northern territories of the Republic of
Karelia are studied relatively poorly from the point of view of economic science and the
socio-economic transformations that have taken place over the past decades. These
circumstances determine the purpose of this study – to update scientific knowledge about
the state and development trends of the human capital in the Karelian Arctic in the context
of the tasks of implementing the special economic regime of the region. In the development of research tools, the works of leading world [5-7] and Russian [8,9]
scientists in the field of human capital research as well as the author's theoretical and
methodological research [10] were implemented. The considerable experience of Russian
researchers in the study of socio-economic processes in the Arctic was also taken into
account [e.g., 11,12]. 3 Results and discussion In the course of the study, 1102 people were interviewed on the territory of the Arctic
Karelia (Kemsky district - 188 people, Loukhsky district - 125 people, Belomorsky district
- 241 people, Kostomuksha urban district - 208 people, Segezha district - 220 people,
Kalevala district - 120 people). Among the respondents, women slightly prevailed: 56.2%
versus 43.8% of men, the average size of households was 2.4 people, among which 61.6%
of families had no underage children. Most of the respondents were at the age of 30-54 -
58.7% (up to 29 years old - 19.9%, 55 years and older - 21.4%), had a secondary special /
vocational (40.7%) or higher (35.3%) education, were employed (73%) or retired (16%). As the results of the study show, the professional activity of a significant number of the
population of Arctic Karelia (44.4%) corresponds to their qualification. At the same time,
most of the respondents who chose the line of work according to their education are located
in the Kemsky district (56.4%). Men more often than women choose a job in accordance with their education (Table 1). Thus, more than half of the male population in the republic (50.6%) work in their field. Among women, there are about 40% of them. This tendency is typical for both the republic
and its regions. The exception was Kalevala district, where the number of women working in their field
of study exceeds the number of men (29.3% versus 26.9%, respectively). On the other
hand, it is in this region that the highest level of non-working men and women is present in
comparison with the average for Arctic Karelia (34.6% and 43.5% versus 27.2% and
14.8%, respectively). A possible explanation for this fact is the assignment of this area to
the regions of the Far North, which means early retirement of the citizens. The results of the correspondence of the work area to the received degree, depending on
the age of the respondents, seem logical. Thus, the maximum number of respondents
working in their field of study is between the ages of 30 and 54 (53.9%). The number of
people employed according to their degree under the age of 29 is slightly less (43.8%). 2 Materials and methods The current research was carried out by a research team in the territory of the Arctic
municipalities of the Republic of Karelia: Kemsky, Belomorsky, Loukhsky, Kalevala and
Segezha municipal districts and Kostomuksha urban district. The informational basis of the
research is the data obtained using the method of mass questionnaire survey of the
population in the territory of Arctic Karelia. The sample is multistage and divided into
districts, with a quota selection of observation units at the last stage. The first stage is
dividing the subjects into districts according to the level of socio-economic development. The second stage – selection of respondents by age and gender quotas, as well as quotas
corresponding to the characteristics of living conditions. Only respondents over 18 years
old were interviewed. The sampling margin of error does not exceed 3%. In addition, the information basis of the research includes regulatory acts of various
administrative levels, statistical data of the Federal State Statistics Service and its territorial
divisions, materials from the websites of local administrations and non-profit organizations. Measurement method: surveying at the place of residence of the respondents. Sample
size: 1102 people aged 18 and over. Technical processing of information was carried out
using the SPSS software. Quantitative indicators for the areas of field research in Arctic
Karelia include Belomorsky, Kemsky, Loukhsky municipal districts, as well as Segezhsky,
Kalevala municipal districts and Kostomuksha urban district, which were included in the
AZRF on July 8 2020. The main methods used in data analysis: quantitative, statistical, including the analysis
of linear distributions, correlation analysis; qualitative, including systematization,
generalization of opinions (assessments). 2 2 https://doi.org/10.1051/e3sconf/202021707028 E3S Web of Conferences 217, 07028 (2020)
ERSME-2020 3 Results and discussion With an increase in age (55 years and older), both the lowest percentage of those working
in their field of study among the other selected groups (18.5%) and the largest number of
non-working citizens (69.5%) were noted. A similar trend is typical for most regions of
Karelia. The exceptions were Kostomuksha urban district and Kalevala district, in which
respondents under the age of 29 work in their degree field more often than those in the age
cohort of 30 to 54 years. y
As the level of income increases, the number of respondents working in their degree
also increases. This tendency was found in all the considered regions of the republic. Assessing the living standards of their families, more than half of the population of
Karelia (51.8%) confirm their ability to pay for their current needs, clothes and food,
however, making larger purchases causes difficulties, and therefore, they are postponed
indefinitely. About 8.4% of respondents, on average, describe their level of income as high,
but women are more modest in this definition. Approximately 6.8% of women indicate this
level of income versus 10.6% of men. The residents of Belomorsk district demonstrate the
highest standard of living (14.1%), although here, too, women are less positive in their
assessments than men. About 9.7% of middle-aged respondents (from 30 to 54 years old) express complete
satisfaction with their own standard of living. Among those who live from paycheck to
paycheck, there is a large number of representatives of the older generation (over 55 years
old). The most positive assessments in relation to their standard of living are shown by the
middle-aged respondents of Belomorsk district (16.7%), while about 6% of the respondents
aged 30 to 54 in Kalevala district state that they do not have enough money for food. 3 3 https://doi.org/10.1051/e3sconf/202021707028 E3S Web of Conferences 217, 07028 (2020)
ERSME-2020 Table 1. The correspondence of the workplace to the received degree, depending on the gender of the
respondents, in percentage of the number of the respondents. 3 Results and discussion District
Gender
Work in
one’s
degree
field
Work in a
related
field
Work in a
different
field
Do not
have a
degree yet
Unemployed
Arctic
Karelia
male
50.6
7.9
22.3
4.4
14.8
female
39.5
7.6
22.5
3.1
27.2
Kemsky
district
male
56.3
21.9
7.8
1.6
12.5
female
56.5
7.3
19.4
1.6
15.3
Loukhsky
district
male
49.2
3.1
18.5
9.2
20.0
female
45.8
1.7
22.0
1.7
28.8
Belomorsky
district
male
56.3
6.8
17.5
4.9
14.6
female
39.1
4.3
25.4
4.3
26.8
Kostomuksha
urban district
male
58.8
6.7
17.6
6.7
10.1
female
25.3
16.1
21.8
8.0
28.7
Segezha
district
male
38.8
5.8
41.7
0.0
13.6
female
37.6
10.3
26.5
0.0
25.6
Kalevala
district
male
26.9
3.8
30.8
3.8
34.6
female
29.3
5.4
18.5
3.3
43.5 Table 1. The correspondence of the workplace to the received degree, depending on the gender of the
respondents, in percentage of the number of the respondents. The answers of the respondents when divided into income groups seem to be quite
logical. About 86% of the wealthier respondents have a sufficient level of income to meet
their current needs. An identical situation emerged according to the results of a survey of
the population in the six studied districts. More often than others, the population of
Belomorsk district indicates that "they live in full prosperity and they do not deny
themselves anything." t e se ves a yt
g. Approximately every second representative of Arctic Karelia (50.6%) positively
assesses the possibility of planning personal income for the year ahead. Every third (29.3%)
resident of Kostomuksha urban district can plan their income for a period up to three years. Planning of personal incomes by men and women on average in the republic does not differ
significantly. Short-term planning up to a year is predominant. The overwhelming majority
of the two groups under consideration (52.2% of men and 49.4% of women) prefer to plan
their income for that period. As the planning period increases, the number of respondents
who prefer to make personal income plans decreases. About 1.2% of citizens consider plans
for more than five years. Interestingly, in Segezha district, about half of the women (47.5%)
and 40% of the men do not plan their personal income at all. Approximately every second representative of Arctic Karelia (50.6%) positively
assesses the possibility of planning personal income for the year ahead. 3 Results and discussion Every third (29.3%)
resident of Kostomuksha urban district can plan their income for a period up to three years. Planning of personal incomes by men and women on average in the republic does not differ
significantly. Short-term planning up to a year is predominant. The overwhelming majority
of the two groups under consideration (52.2% of men and 49.4% of women) prefer to plan
their income for that period. As the planning period increases, the number of respondents
who prefer to make personal income plans decreases. About 1.2% of citizens consider plans Young people under the age of 29 plan personal incomes most often. Over a third of the
respondents over 55 years old do not plan personal income at all. In Loukhsky, Segezha and
Kalevala districts, the tendency is somewhat different: it is mostly the middle-aged citizens
(30-54 years old) who prefer to plan their personal income for up to a year. About half of the respondents (45.3%) with a low income level prefer not to plan their
personal income at all. High-income citizens are more active in long-term planning: about
20% make plans for up to three years. In Loukhsky, Belomorsky districs and Kostomuksha
urban district of the republic, a significant number (above the national average) of the
respondents with a low income level were recorded, who prefer short-term planning up to a
year. y
Only a small number of the respondents can plan their family's income for a period of
more than five years. Short-term income plans for one year are most in demand by the
women (62.7%) and the men (57.6%) of Loukhsky district. In different age groups, family income planning does not differ significantly from
personal income planning. As the obtained data show, as the age of the respondents 4 4 E3S Web of Conferences 217, 07028 (2020)
ERSME-2020 https://doi.org/10.1051/e3sconf/202021707028 increases, the desire/ability to plan family income for a five-year period is an insignificant
percentage. Interestingly, about 84.4% of the men in Segezha district generally respond
negatively to the question about the possibilities of planning their family's income. increases, the desire/ability to plan family income for a five-year period is an insignificant
percentage. Interestingly, about 84.4% of the men in Segezha district generally respond
negatively to the question about the possibilities of planning their family's income. 4 Conclusion In summary, the results of the survey conducted allow us to draw the following
conclusions: 1. The population of Arctic Karelia for the most part works in the field of the
previously received education. A more pragmatic choice of work in accordance with the
received degree is typical for the male population, as well as for persons aged 30 to 54 and
with a higher level of income. 1. The population of Arctic Karelia for the most part works in the field of the
previously received education. A more pragmatic choice of work in accordance with the
received degree is typical for the male population, as well as for persons aged 30 to 54 and
with a higher level of income. 2. The majority of the population of the republic considers their living standards as
sufficient only to pay for current needs. Inhabitants of Belomorsk district assess their living
standards more positively. Women are generally more modest in their assessments of their
standard of living than men. Middle-aged people are more optimistic about their standard of
living, describing it as “we live in full prosperity, we do not deny ourselves anything”. 3. Planning of personal incomes, as well as incomes of their families, by the population
of the republic is mostly for a short term. The overwhelming majority of the respondents
note the possibility to make plans for their income for a period of no more than a year. Every third resident does not plan their personal income, and a small number of citizens
(1.2%) have the opportunity to plan income for a longer period (more than five years). Men
are characterized by a longer planning period for their personal incomes compared to
women. Representatives of the middle age category, as well as those with a high level of
income, have the opportunity to plan their income for a longer period. Further research involves the analysis of factual data collected during fieldwork on the
following topical issues of human capital development in the Karelian Arctic: Further research involves the analysis of factual data collected during fieldwork on the
following topical issues of human capital development in the Karelian Arctic: 1. Analysis of the sources of income and material well-being of the population in the
context of age and gender groups 2. Existing practices of increasing the human capital among the population by gender
and age groups. 3 Results and discussion Short-term planning of income (up to 1 year) is typical for both high-income categories
of the population and people with a low level of material well-being. A planning period of
more than a year is most in demand in a high-income stratum of the population. A
significant number of citizens with a low level of income in all the districts of the republic
under consideration are characterized by a lack of opportunities for planning their family
income. 4 Conclusion The study was conducted within the framework of the state assignment of the Ministry of Science and
Higher Education of the Russian Federation "Institutions and social inequality in the context of global
challenges and regional restrictions (0218-2019-0090)". 1. Decree of the President of the Russian Federation "On the Strategy for the
Development of the Arctic Zone of the Russian Federation and Ensuring National
Security for the Period until 2035" No. 645 of October 26, 2020. Available from:
http://www.kremlin.ru/acts/news/64274 [Accessed 29th October 2020] References 1. Decree of the President of the Russian Federation "On the Strategy for the
Development of the Arctic Zone of the Russian Federation and Ensuring National
Security for the Period until 2035" No. 645 of October 26, 2020. Available from:
http://www.kremlin.ru/acts/news/64274 [Accessed 29th October 2020] 1. Decree of the President of the Russian Federation "On the Strategy for the
Development of the Arctic Zone of the Russian Federation and Ensuring National
Security for the Period until 2035" No. 645 of October 26, 2020. Available from:
http://www.kremlin.ru/acts/news/64274 [Accessed 29th October 2020] 5 https://doi.org/10.1051/e3sconf/202021707028 E3S Web of Conferences 217, 07028 (2020)
ERSME-2020 2. V. Tsukerman, E. Goryachevskaya, S. Ivanov, IOP Conference Series: Earth and
Environmental Science 302, 012109 (2019) https://doi.org/10.1088/1755-
1315/302/1/012110 3. Decree of the President of the Russian Federation of June 27, 2017 No. 287 "On
amendments to the Decree of the President of the Russian Federation of May 2, 2014
No. 296 "On the land territories of the Arctic zone of the Russian Federation"
Available from: http://www.kremlin.ru/acts/bank/42021 [Accessed 29th October 2020] 4. Federal Law of July 13, 2020 N 193-FL "On state support for entrepreneurial activity
in
the
Arctic
zone
of
the
Russian
Federation"
Available
from:
http://publication.pravo.gov.ru/Document/View/0001202007130047 [Accessed 29th
October 2020] 5. M. Blaug, Methodology of Economic Science, or How Economists Explain (Moscow:
NP "Journal of Economic Issues", 2004) 6. B. Égert, J. Botev, D. Turner, Eur. Econ. Rev. 129, 103560 (2020)
https://doi.org/10.1016/j.euroecorev.2020.103560. 7. O. Attanasio, Journal of the European Economic Association 13, 6, 1, 949–997
(2015) https://doi.org/10.1111/jeea.12159 8. A. Koritskiy, Human capital as a factor of economic growth in Russian regions: a
monograph (Nov. Sib. Univ. of Cons. Coop., 2010) 9. V. Samarina, T. Skufina, A. Samarin, European Research Studies Journal 21, 3, 705–
716 (2018) https://doi.org/10.35808/ersj/1094 10. A. Volkov, G. Kozyreva, A. Morozov, Economic systems management 11 (2017) 11. V. Tatarkin, V. Litovskiy, MSTU Bulletin 3, 573-587 (2014) 12. S. Baranov, T. Skuf'ina, I. Gushchina, Economic and social changes: facts, trends,
forecast 1, 160-173 (2020) 6 6
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ADRB1 was identified as a potential biomarker for breast cancer by the co-analysis of tumor mutational burden and immune infiltration
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AGING 2021, Vol. 13, No. 1 AGING 2021, Vol. 13, No. 1 www.aging-us.com Research Paper
ADRB1 was identified as a potential biomarker for breast cancer by
the co-analysis of tumor mutational burden and immune infiltration
Jia Wang1, Xiaolu Zhang2, Jie Li1, Xiaoran Ma2, Fubin Feng3, Lijuan Liu3, Jibiao Wu1, Changgang
Sun3,4
1College of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250014,
Shandong, P. R. China
2College of First Clinical Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong,
P. R. China
3Department of Oncology, Weifang Traditional Chinese Hospital, Weifang 261000, Shandong, P. R. China
4Innovative Institute of Chinese Medicine and Pharmacy, Shandong University of Traditional Chinese Medicine,
Jinan 250014, Shandong, China
Correspondence to: Changgang Sun; email: scgdoctor@126.com, https://orcid.org/0000-0002-6648-3602
Keywords: breast cancer, tumor mutational burden, immune infiltration, ADRB1, prognosis
Received: August 6, 2020
Accepted: September 29, 2020
Published: November 21, 2020
Copyright: © 2020 Wang et al. This is an open access article distributed under the terms of the Creative Commons
Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. Research Paper
ADRB1 was identified as a potential biomarker for breast cancer by
the co-analysis of tumor mutational burden and immune infiltration
Jia Wang1, Xiaolu Zhang2, Jie Li1, Xiaoran Ma2, Fubin Feng3, Lijuan Liu3, Jibiao Wu1, Changgang
Sun3,4 Jia Wang1, Xiaolu Zhang2, Jie Li1, Xiaoran Ma2, Fubin Feng3, Lijuan Liu3, Jibiao Wu1, Changgang
Sun3,4 Correspondence to: Changgang Sun; email: scgdoctor@126.com, https://orcid.org/0000-0002-6648-3602
Keywords: breast cancer, tumor mutational burden, immune infiltration, ADRB1, prognosis
Received: August 6, 2020
Accepted: September 29, 2020
Published: November 21, 2020 Copyright: © 2020 Wang et al. This is an open access article distributed under the terms of the Creative Commons
Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. ABSTRACT Breast cancer (BRCA) has traditionally been considered as having poor immunogenicity and is characterized by
relatively low tumor mutational burden (TMB). Improving immunogenicity may improve the response to clinical
immunotherapy of BRCA. However, the relationship between TMB, immune infiltration, and prognosis in BRCA
remains unclear. We aimed to explore their interrelations and potential biomarkers. In this study, based on
somatic mutation data of BRCA from The Cancer Genome Atlas (TCGA), patients were categorized into high and
low TMB groups utilizing the TMB values. CIBERSOFT algorithm indicated significant infiltration of activated
partial immune cells in high TMB group. Besides, ADRB1 had been identified as a prognosis-related immune
gene in the mutant genes by the combination of the ImmPort database and the univariate Cox analysis. ADRB1
mutation was associated with lower TMB and manifested a satisfactory clinical prognosis. Various database
applications (Gene Set Enrichment Analysis, Tumor IMmune Estimation Resource, Connectivity Map, KnockTF)
supported the selection of treatment strategies targeting ADRB1. In conclusion, TMB was not an independent
prognostic factor for BRCA and high TMB was more likely to activate a partial immune response. ADRB1 was
identified as a potential biomarker and may provide new insights for co-therapy of BRCA. Somatic mutation landscape in BRCA BRCA has traditionally been considered as having poor
immunogenicity and is characterized by relatively low
TMB [11]. However, the immune responses vary
substantially between BRCA subtypes. Triple negative
breast cancer (TNBC) and HER-2 (+) BRCA are
generally more immunogenic than hormone-sensitive
BRCA, as reflected in a higher proportion of tumor
infiltrating lymphocytes [12]. In addition, luminal B
subtypes can be more immunogenic than luminal A
tumors among hormone-sensitive BRCA [13]. Allison
and Vogelstein have reported a large number of new
antigens in breast and bowel cancer tissues, and all
cancers have the potential to accumulate new antigens
that the immune system can recognize during
tumorigenesis [14]. These findings suggest that TMB
may play a predictive role in BRCA. Studies have
demonstrated that the proliferation rate and the intrinsic
subtype of BRCA were associated with TMB [15, 16],
whose role in tumor immunogenicity in BRCA is still
unclear. Analysis of the 1,044 BRCA mutation samples from
TCGA is shown in Figure 1A. Missense mutation was
the primary variant classification and all mutations
belonged to single nucleotide polymorphisms. C>T was
the most common variation in BRCA with the highest
number of variations per sample and the median of
variation types. In addition, the frequencies of
mutations in PIK3CA (29%) and TP53 (27%) were the
highest in mutant genes, all of which were missense
mutations (Figure 1B). MUC17, HUWE1, SYNE1,
TTN, MUC16, HMCN1 had equally higher co-mutation
frequencies, while CDH1 and TP53 showed obvious
mutuality of mutual exclusion (Figure 1C). Correlation analysis of TMB The TMB in BRCA ranged from 0.02 to 112.8 per Mb
with a median of 0.86 per Mb. With the median TMB
value set as the threshold, a total of 986 samples was
divided into the high (n=493) and low TMB (n=493)
groups. We performed Kaplan-Meier analysis and
determined that the 5-year survival rate of was 0.774 for
the high TMB group and 0.870 for the low TMB group. Since the high TMB group predicted a better prognosis
beyond 10 years, TMB may not be an independent
prognostic factor for BRCA (Figure 2A). In addition,
among six clinical characteristics, only age and the N
stage
were
significantly
correlated
with
TMB;
specifically, patients over 65 years old or with
uninvolved regional lymph nodes had higher TMB
(Figure 2B). The differential expression of 454 mutant
genes between groups is shown in Figure 2C. Meanwhile, certain issues remain with TMB. Previous
clinical research found that comparing to patients in
the low TMB group, not all patients in the high TMB
group benefited from ICIs. Specifically, a subset of
patients with mutations in the ERBB family (EGFR
/ERBB2) and the deletion of specific 3p segments of
the chromosome did not respond to ICIs [17]. Cristescu et al. published a study in Science [18],
which has some implications for us: simultaneous
detection of T cell activity levels and TMB may be a
promising strategy. Indeed, positive correlations
between mutations or new antigen loads and immune
infiltration have been observed in various cancer types
[19, 20]. Therefore, we hypothesized that in
combination with immune cell groups, TMB as a
quantitative indicator of tumor antigenicity may
influence the prognosis of BRCA. RESULTS urothelial carcinoma, non-small-cell carcinoma, and
bladder cancer [7–10]. INTRODUCTION [3, 4]. It is worth mentioning that TMB may be a
promising tumor biomarker [5], defined as the total
number
of somatic
gene
coding errors,
base
substitutions, gene insertions or deletion errors per
megabase (Mb). Higher TMB in tumors was reported
to facilitate the formation of more new antigens and
enhance tumor immunogenicity, which could improve
clinical responses to cancer immunotherapy [6]. For
example, patients with high TMB had better responses
to ICIs and improved survival rates in melanoma, Programmed death-1 (PD-1) and programmed death
ligand-1 (PD-L1) are immune checkpoint inhibitors
(ICIs) [1], which is the most studied type of
immunotherapy for breast cancer (BRCA) according to
relevant statistics [2]. TMB is a novel marker for
evaluating the therapeutic effect of PD-1 antibodies,
which has been confirmed in the treatment of
colorectal cancer with defects in mismatch repair AGING 351 www.aging-us.com Relationship between TMB and immune infiltration The CIBERSOFT algorithm was used to assess the
abundance of immune cells in the high and low TMB
groups, and to explore the intrinsic relationship between
TMB and the survival rate. Compared to those in the
low TMB group (Figure 3A), there were lower levels of
B cells and T cells, and higher levels of macrophages in
the high TMB group (Figure 3B). Further comparisons
indicated that naive/memory B cells, resting CD4+
memory T cells, follicular helper T cells, gamma delta
T cells, resting dendritic cells, and resting mast cells
were abundant in the low TMB group (Figure 3C). For
the high TMB group, there were significant infiltration
of
activated
CD4+
memory
T
cells,
M0/M1
macrophages,
and
activated
dendritic
cells. Furthermore, there were expressional correlations
among the subsets of immune cells in transcriptome,
a
significant
negative
correlation
between
M0 In this study, we investigated the association of TMB
with gene mutations, immune responses, and prognosis
of BRCA in combination with tumor immune
infiltration. Using the gene expression profiling data of
BRCA from the TCGA database, different gene
expressions between high and low TMB groups were
compared, and aspects of the clinical characteristics,
gene functions and pathways, as well as immune
responses were further evaluated. We attempted to
elucidate these relationships: different TMB and clinical
outcomes, TMB and immune cell populations, immune
cells affected by TMB and prognosis. The findings of
this study may provide new biomarkers and potential
therapy options for BRCA in the future. AGING 352 www.aging-us.com (Figure 4A), ADRB1 was significantly enriched in G-
protein coupled receptor binding, neurotransmitter
receptor activity, neurotransmitter receptor activity
involved in the regulation of postsynaptic membrane
potential, and postsynaptic neurotransmitter receptor
activity in molecular function, regulation of membrane
potential, positive regulation of heart contraction, and
heat generation in biological process, synaptic membrane
and postsynaptic membrane in cellular component. Gene
Set Enrichment Analysis (GSEA) performed with TCGA
data indicated that the calcium signaling pathway, dilated
cardiomyopathy, endocytosis, and neuroactive ligand-
receptor
interactions
were
significantly
enriched macrophages and resting CD4+ memory T cells,
whereas activated CD8+ and CD4+ memory T cells were
positively correlated (Figure 3D). The Venn diagram
showed that 44 immune genes in the differentially
expressed genes (DEGs) were screened out (Figure 3E)
and ADRB1 was identified as a prognosis-related
immune gene by the univariate Cox regression analysis
(Table 1). Functional enrichment analysis We further examined the functional enrichment of DEGs
especially ADRB1. Based on gene ontology categories Figure 1. The landscape of mutation genes in BRCA samples. (A) Classification of mutation types according to different categories, in
which missense mutation accounts for the most fraction; SNP appears in all mutations; and C>T is the most common SNV; tumor mutational
burden in specific samples; the top 10 mutated genes in BC. (B) Mutation information of each gene in each sample is shown in the waterfall
plot, in which various colors with annotations at the bottom represent the different mutation types. The bar plot above the legend shows the
tumor mutational burden; (C) The coincident and exclusive associations across mutated genes. SNP, single nucleotide polymorphism; SNV,
single nucleotide variants; BRCA, breast cancer. Figure 1. The landscape of mutation genes in BRCA samples. (A) Classification of mutation types according to different categories, in
which missense mutation accounts for the most fraction; SNP appears in all mutations; and C>T is the most common SNV; tumor mutational
burden in specific samples; the top 10 mutated genes in BC. (B) Mutation information of each gene in each sample is shown in the waterfall
plot, in which various colors with annotations at the bottom represent the different mutation types. The bar plot above the legend shows the
tumor mutational burden; (C) The coincident and exclusive associations across mutated genes. SNP, single nucleotide polymorphism; SNV,
single nucleotide variants; BRCA, breast cancer. Figure 1. The landscape of mutation genes in BRCA samples. (A) Classification of mutation types according to different categories, in
which missense mutation accounts for the most fraction; SNP appears in all mutations; and C>T is the most common SNV; tumor mutational
burden in specific samples; the top 10 mutated genes in BC. (B) Mutation information of each gene in each sample is shown in the waterfall
plot, in which various colors with annotations at the bottom represent the different mutation types. The bar plot above the legend shows the
tumor mutational burden; (C) The coincident and exclusive associations across mutated genes. SNP, single nucleotide polymorphism; SNV,
single nucleotide variants; BRCA, breast cancer. AGING 353 www.aging-us.com (p<0.001). In addition, a high level of B cells suggested
good prognosis of BRCA, and high expression of
ADRB1 may prompt better survival (Figure 5B). in samples with ADRB1 (Figure 4B–4E). Functional enrichment analysis The findings
also showed that the four pathways ADRB1 located in
were all significantly active in the low-TMB group. Various small-molecule drugs of ADRB1 and the
transcription factor HIF1A CNV of ADRB1, immune cells, and survival in
BRCA Among the 43 targeted small-molecule drugs predicted
for ADRB1, 17 were identified to be acting on BRCA-
associated
genes
(Table
2),
including
vascular
endothelial growth factor A (VEGFR1), dopamine
receptor,
prolactin,
tumor
necrosis
factor,
and
polycyclic aromatic hydrocarbons. In the hypoxia Generally, copy number variations (CNVs) refers to the
increase or decrease in the copy number of a large
segment in the genome whose length exceeds 1 kb. The
results were presented in Figure 5A. In B cells and
dendritic cells, high amplification of ADRB1 was
significantly different compared to other CNVs Figure 2. Performance evaluation of TMB and DEGs in the high and low TMB groups. (A) Prognosis of TMB. The survival curves of
the high and low TMB groups intersect (P=0.022); (B) The associations of the clinical characteristics with TMB. Higher TMB levels were
associated with over 65 years old and the N0 stage (P<0.001); (C) The top 40 DEGs are shown in the heatmap plot. TMB, tumor mutation
burden; DEGs, differentially expressed genes; N0, no lymph nodes are involved. Figure 2. Performance evaluation of TMB and DEGs in the high and low TMB groups. (A) Prognosis of TMB. The survival curves of
the high and low TMB groups intersect (P=0.022); (B) The associations of the clinical characteristics with TMB. Higher TMB levels were
associated with over 65 years old and the N0 stage (P<0.001); (C) The top 40 DEGs are shown in the heatmap plot. TMB, tumor mutation
burden; DEGs, differentially expressed genes; N0, no lymph nodes are involved. AGING 354 www.aging-us.com follicular helper T cells, gamma delta T cells, and
various resting immune cells. According to a recent
study on triple-negative BRCA [22], the research team
used a corresponding single anti CD8+ T cells in
immune treatment to activate related anti-tumor
immune mechanism, while ICIs activated follicular
helper T cells that stimulated B cells to produce
antibodies. However, the impact on tumor immune
responses in inhibiting follicular helper T cells and B
cells were more profound than inhibiting CD8+ T cells,
which demonstrates that B cells and follicular helper T
cells play key roles in tumor immune responses. Moreover, higher levels of gamma delta T cells have
been shown to be correlated with better outcomes [23]. Various small-molecule drugs of ADRB1 and the
transcription factor HIF1A On the other hand, tumor immunogenicity was
enhanced in the high TMB group, leading to significant
infiltration of CD4+ memory T cells, M0/M1
macrophages, and dendritic cells as well as activated
immune responses. The relative increase in TMB was
also associated with aging and the N stage, consistent
with previous literature that mutations of TP53 in lymph
node-negative BRCA were higher than those in lymph
node-positive BRCA, and mutations in microtubule-
associated proteins may help immune cells recognize
tumors and inhibit lymph node metastasis [24]. inducible factor A (HIF1A) knocking-down dataset in
MCF-7 cells (Table 3), the upregulation of ADRB1
maybe not directly regulated by HIF1A, and it might be
combined with the AFF1 factor to cause the knock-on
effects, AFF1 was detected to bind to the super enhancer
and typical enhancer region of the target gene (ADRB1),
the regulatory mechanism within is unclear. However, we
accidently found that the PIK3CA gene (Figure 6) was
involved in the VEGFR1-specific signaling pathway that
HIF1A participates in, which has the highest proportion
of gene mutations in this study. Its role needs to be
further studied. DISCUSSION TMB was calculated based on the BRCA mutation data
from TCGA, and the relationship between the survival
curve and TMB showed that TMB may not be an
independent prognostic factor for BRCA, which is
consistent with previous studies on HER2 (-) metastatic
BRCA [21]. We speculated that TMB combined with
other prognostic factors may have a better predictive
effect. To clarify the internal relationship between TMB
and immunologic infiltration, we further showed that
the low TMB group had abundant levels of B cells, Figure 3. Immune cell content in the high and low TMB groups and the identification of TMB-related immune genes. (A, B)
The stacked bar chart indicates the distribution of 22 immune cells in the low and high TMB groups, respectively; (C) The violin plot indicates
the differentially infiltrated immune cells between in the high and low TMB groups. The green color represents the low TMB group, and the
red color represents the high TMB group; (D) The correlation matrix of immune cell proportions. The red color represents positive
correlations and the blue color represents negative correlations; (E) The identification of TMB-related immune genes. Figure 3. Immune cell content in the high and low TMB groups and the identification of TMB-related immune genes. (A, B)
The stacked bar chart indicates the distribution of 22 immune cells in the low and high TMB groups, respectively; (C) The violin plot indicates
the differentially infiltrated immune cells between in the high and low TMB groups. The green color represents the low TMB group, and the
red color represents the high TMB group; (D) The correlation matrix of immune cell proportions. The red color represents positive
correlations and the blue color represents negative correlations; (E) The identification of TMB-related immune genes. AGING 355 www.aging-us.com Table 1. Identification of TMB-related immune genes and the univariate Cox regression analysis in BRCA. DISCUSSION Gene
HR
HR.95L
HR.95H
CoxPvalue
ADRB1*
0.824
0.688
0.987
0.035
SEMA6D*
1.041
1.009
1.074
0.011
FGF14*
1.052
1.006
1.101
0.025
SCG2
1.004
1.002
1.006
9.936
CXCL14
0.999
0.999
1.000
0.320
TMSB15A
1.006
0.992
1.020
0.352
UMODL1
0.966
0.766
1.219
0.775
TNFSF11
0.992
0.951
1.035
0.742
CHGA
0.997
0.990
1.004
0.528
RLN2
1.001
0.986
1.015
0.892
STC2
1.000
0.999
1.001
0.787
TAC1
0.894
0.677
1.179
0.428
ULBP1
1.113
0.977
1.268
0.104
RAET1L
1.074
0.984
1.173
0.107
PDIA2
0.988
0.784
1.274
0.924
SLPI
1.000
0.999
1.000
0.807
LCN2
0.999
0.998
1.001
0.984
S100A9
0.999
0.999
1.000
0.586
S100A8
0.999
0.999
1.000
0.740
MMP12
1.003
0.985
1.020
0.722
PGLYRP4
1.036
0.874
1.229
0.677
FABP6
1.034
0.984
1.087
0.178
MUC5AC
1.003
0.998
1.007
0.145
MARCO
0.992
0.974
1.011
0.430
PCSK1
1.000
0.999
1.001
0.470
VTN
1.003
0.991
1.015
0.557
CCL14
0.898
0.722
1.117
0.336
CMA1
0.995
0.852
1.163
0.958
FGF10
1.001
0.995
1.008
0.641
CX3CR1
0.987
0.941
1.034
0.589
CHGB
1.000
0.999
1.000
0.080
EPO
1.007
0.978
1.037
0.594
GDNF
1.007
0.981
1.032
0.589
GHRH
0.992
0.939
1.047
0.781
NRTN
1.000
0.942
1.062
0.987
NTS
0.988
0.965
1.011
0.307
PTHLH
0.996
0.985
1.008
0.611
SLURP1
1.020
0.993
1.049
0.136
CRLF1
0.994
0.976
1.013
0.557
FGFR4
1.010
0.993
1.027
0.216
IL12RB2
0.955
0.844
1.081
0.472
IL1RL1
1.012
0.909
1.127
0.816
IL22RA2
0.758
0.536
1.071
0.116
PGR
0.996
0.985
1.008
0.583
*, coxPvalue< 0.05. Furthermore, correlations within the immune cells were
determined by analyzing the immune matrix of the
entire transcriptome. When investigating the clinical
significance of infiltrated immune cells in BRCA, the
higher proportion of M0 macrophages indicated a
reduced disease-free survival, whereas the increased Furthermore, correlations within the immune cells were
determined by analyzing the immune matrix of the
entire transcriptome. When investigating the clinical
significance of infiltrated immune cells in BRCA, the
higher proportion of M0 macrophages indicated a
reduced disease-free survival, whereas the increased overall survival was associated with a relatively higher
resting CD4+ memory T cells score [25], which
corroborates the results of this study. In general,
differences in immunogenicity may lead to differences
in the activation of immune mechanisms, and the few
types of immune cell activation in the high TMB group AGING 356 www.aging-us.com low level of mutant allele tumor heterogeneity [26],
confirming the correlation between TP53 and CDH1
observed in this study. DISCUSSION The PIK3CA mutation was
found in different subtypes such as ER (+), PR (+),
HER2 (+), and TNBCs [27, 28], but its role in
VEGFR1-specific signaling pathway needs to be further might indicate that higher TMB suppresses the immune
response. It is also worthwhile to note that PIK3CA and
TP53 had prominent performance among mutation
genes. Previous studies have revealed that mutant allele
tumor heterogeneity is positively correlated with TP53
mutation rate, while CDH1 mutation is correlated with a Figure 4. Functional enrichment analysis. (A) MF, BP, CC in GO categories of DEGs; (B–E) ADRB1 related pathways using the GSEA
software. MF, molecular functions; BP, biological processes; CC, cellular components; GO, gene ontology; DEGs, differentially expressed
genes. Figure 4. Functional enrichment analysis. (A) MF, BP, CC in GO categories of DEGs; (B–E) ADRB1 related pathways using the GSEA
software. MF, molecular functions; BP, biological processes; CC, cellular components; GO, gene ontology; DEGs, differentially expressed
genes. Figure 5. Correlations between the CNV of ADRB1, immune cell infiltration, and prognosis. (A) High amplification of ADRB1 in B
cells and dendritic cells (p<0.001); (B) High levels of B cells and ADRB1 suggested better prognosis of BRCA (p<0.05). CNV, copy number
variations; BRCA, breast cancer. Figure 5. Correlations between the CNV of ADRB1, immune cell infiltration, and prognosis. (A) High amplification of ADRB1 in B
cells and dendritic cells (p<0.001); (B) High levels of B cells and ADRB1 suggested better prognosis of BRCA (p<0.05). CNV, copy number
variations; BRCA, breast cancer. AGING 357 www.aging-us.com www.aging-us.com
358
AGING
Table 2. Seventeen small-molecule drugs predicted by ADRB1 as well as their effects on BRCA-associated genes. DISCUSSION Seventeen small-molecule drugs predicted by ADRB1 as well as their effects on BRCA-assoc Adrenergic receptor
antagonist, Prolactin inhibitor
Dopamine receptor antagonist,
Dopamine receptor ligand,
Serotonin receptor antagonist DISCUSSION Name
Target
MOA
amiodarone
KCNH2, ADRB1, CACNA1H, CACNA2D2, CHRM3, CYP2C8,
KCNA7, SCN5A
Potassium channel blocker
carvedilol
ADRB1, ADRB2, ADRA1A, ADRA1B, ADRA1D, ADRA2A,
ADRA2B, ADRA2C, ADRB3, CYP2C19, CYP2E1, GJA1, HIF1A,
KCNH2, NDUFC2, NPPB, RYR2, SELE, VCAM1, VEGFA
Adrenergic receptor antagonist
desipramine
SLC6A2, SLC6A4, ADRA1A, ADRA1B, ADRA1D, ADRA2A,
ADRA2B, ADRA2C, ADRB1, ADRB2, CHRM1, CHRM2,
CHRM3, CHRM4, CHRM5, DRD2, HRH1, HTR1A, HTR2A,
HTR2C, SMPD1
Tricyclic antidepressant
dihydroergocristine
HTR2A, ADRA1A, ADRB1, DRD1, DRD2, DRD3, DRD4, DRD5,
HTR1A, HTR3A, HTR4, HTR5A, HTR6, HTR7
Adrenergic receptor
antagonist, Prolactin inhibitor
loxapine
DRD2, DRD3, DRD4, DRD1, HRH1, HTR2A, HTR2C, HTR6,
ADRA1A, ADRA1B, ADRA2A, ADRA2B, ADRA2C, ADRB1,
CHRM1, CHRM2, CHRM3, CHRM4, CHRM5, DRD5, HRH2,
HRH4, HTR1A, HTR1B, HTR1D, HTR1E, HTR3A, HTR5A,
HTR7, SLC6A2, SLC6A3, SLC6A4
Dopamine receptor antagonist,
Dopamine receptor ligand,
Serotonin receptor antagonist
mirtazapine
ADRA2A, HTR2A, HTR2C, ADRA2C, HTR3A, ADRA1A,
ADRA1B, ADRA1D, ADRA2B, ADRB1, ADRB2, DRD1, DRD2,
DRD3, DRD5, HRH1, HRH3, HTR2B, HTR7, OPRK1, SLC6A2,
SLC6A3, SLC6A4
Adrenergic receptor
antagonist, Serotonin receptor
antagonist
sotalol
ADRB1, ADRB2, KCNH2
Adrenergic receptor antagonist
trimipramine
SLC6A2, SLC6A4, SLC6A3, ADRA1A, ADRA1B, ADRA2A,
ADRA2B, ADRB1, ADRB2, ADRB3, CHRM1, CHRM2, CHRM3,
CHRM4, CHRM5, DRD1, DRD2, DRD5, HRH1, HTR1A, HTR1D,
HTR2A, HTR2C, HTR3A
Norepinephrine reuptake
inhibitor, Tricyclic
antidepressant
amitriptyline
CHRM1, CHRM2, CHRM3, CHRM4, CHRM5, HRH1, HTR6,
SLC6A2, SLC6A4, ADRA1A, ADRA1B, ADRA1D, ADRA2A,
ADRB1, ADRB2, ADRB3, HRH2, HRH4, HTR1A, HTR1B,
HTR1D, HTR2A, HTR2C, HTR7, KCNA1, KCND2, KCND3,
KCNQ2, KCNQ3, NTRK1, NTRK2, OPRD1, OPRK1, OPRM1,
SIGMAR1
Norepinephrine inhibitor,
Norepinephrine reuptake
inhibitor, Serotonin receptor
antagonist, Serotonin reuptake
inhibitor
cabergoline
DRD2, ADRA1A, ADRA2A, ADRA2B, ADRA2C, DRD1, DRD3,
DRD4, DRD5, HTR1A, HTR1B, HTR1D, HTR2A, HTR2B,
HTR2C, ADRA1B, ADRA1D, ADRB1, ADRB2, HTR7, PRL
Dopamine receptor agonist
nortriptyline
KCNJ10, SLC6A2, SLC6A4, ADRA1A, ADRA1B, ADRA1D,
ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3,
CHRM1, CHRM2, CHRM3, CHRM4, CHRM5, CYP2C19, DRD2,
HRH1, HTR1A, HTR2A, HTR2C, HTR6, PGRMC1, PIK3CD,
SIGMAR1
Tricyclic antidepressant
propafenone
KCNH2, SCN5A, ADRB1, ADRB2, KCNA5, KCNK2, KCNK3
Antiarrhythmic
pseudoephedrine
ADRA1A, ADRA2A, ADRB1, ADRB2, ATF1, ATF2, ATF3,
ATF4, ATF5, ATF6, ATF7, CXCL8, FOS, HRH1, IL2, JDP2, JUN,
NFATC1, SLC6A2, SLC6A3, SLC6A4, TNF
Adrenergic receptor agonist
propranolol
ADRB2, ADRB3, ADRB1, CYP2C19, HTR1A, HTR1B
Adrenergic receptor antagonist
olanzapine
DRD2, HTR2A, HTR2C, DRD1, DRD3, DRD4, HRH1, HTR1A,
HTR1B, HTR1D, HTR1E, HTR6, HTR7, ADRA1A, ADRA1B,
ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3,
CHRM1, CHRM2, CHRM3, CHRM4, CHRM5, CYP2C8, DRD5,
GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6,
GABRB1, GABRB2, GABRB3, GABRD, GABRE, GABRG1,
GABRG2, GABRG3, GABRP, GABRQ, HRH2, HRH4, HTR1F,
HTR2B, HTR3A, HTR5A
Dopamine receptor antagonist,
Serotonin receptor antagonist
epinephrine
ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C,
ADRB1, ADRB2, ADRB3, PAH, TNF
carbonic anhydrase activator ecule drugs predicted by ADRB1 as well as their effects on BRCA-associated genes. SIGMAR1 AGING 358 www.aging-us.com norepinephrine
ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C,
ADRB1, ADRB3, ADRB2, DRD1, DRD5, PAH, SLC18A1,
SLC18A2
Adrenergic receptor agonist norepinephrine Table 3. Transcription factors regulating ADRB1 in the breast tissue. Table 3. Transcription factors regulating ADRB1 in the breast tissue. Target gene
TF
Knock-method
Tissue type
Biosample name
LogFC
Corrected_P
ADRB1
ELK3
shRNA
Mammary_gland
MDA-MB231
0.68636
5.70000e-04
PTEN
shRNA
SKBR3
0.89431
6.21560e-01
XBP1
shRNA
MDA-MB231
0.90654
1.24800e-02
shRNA
T47D
0.96355
1.65000e-03
HIF1A
siRNA
MCF7
1.09792
6.49070e-01
TF, transcription factor. explored. Patients with somatic mutations in TP53 and
PIK3CA had reported poor survival [29], and whether
the co-mutation of TP53 and PIK3CA can be potential
biomarkers for different subtypes of BRCA warrants
further investigation. activated by ADRB1 phosphorylates troponin I, the L-
type
Ca2+
channel
and
phospholamban,
while
increasing cardiac inotropy, chronotropy, and work
[30]. In
neuroinflammatory
diseases,
ADRB1
activation may have neuroprotective effects [31]. Furthermore, experiments have revealed that AR
signaling can stimulate the transformation of epithelial
cells to mesenchymal cells [32], and ADRB1 was
observed to be overexpressed in BRCA tissues [33]. High
expression levels of ADRB1 can predict better prognosis
in this study, possibly because the overexpression of AR
enhances the sensitivity of the tumor to β-blockers,
although a previous report claimed that there was no
correlation [34]. Future research should further clarify this
issue. Pharmacoepidemiologic studies have shown that β-
blockers could reduce disease progression and mortality
by inhibiting the metastasizing effect of AR signaling
[35], but a retrospective analysis indicated that selective
β-blockers alone or in combination were less effective
than
non-selective
β-blockers
in
reducing
cell
proliferation in BRCA [33]. Interestingly, long-term
deprivation of ovarian sex hormones can induce the
upregulation of ADRB1 in the heart of rats [36], which
suggested that the expression of ADRB1 is up-regulated
when the sex hormone shows negative, and in our study,
high expression of ADRB1 predicts better prognosis. Therefore, we speculate that HER2 (+) and triple-negative
BRCA may be sensitive to β-blockers comparing to sex
hormone types (such as the luminal subtype). That is,
these two types of breast cancer may be easier to benefit
from co-therapy. Prospective clinical trials of β-blockers
on various subtypes of BRCA should be the focus of
future research. ADRB1 was eventually identified as a prognosis-related
immune gene for BRCA, whose functions were further
explored. TMB grouping and differential expression analysis Normal samples in the tumor mutation data were deleted
and the remaining tumor samples were cross-analyzed
with the transcriptome samples. The median value of
TMB was used as the threshold to divide samples into
high and low TMB groups. The DEGs between the two
groups were identified using the Wilcoxon rank test. The
p value was adjusted by the false discovery rate (FDR) to
improve the accuracy of the results, and the thresholds
were set as FDR < 0.05 and logFC (fold change) > 1.0. CNV and immune cells Tumor IMmune Estimation Resource (TIMER v2.0,
https://cistrome.shinyapps.io/timer/), a web server for
comprehensive analysis of tumor-infiltrating immune
cells, was used to estimate the abundances of six
immune infiltrates (B cells, CD4+ T cells, CD8+ T cells,
neutrophils, macrophages, and dendritic cells) [39]. Changes in CNV were observed in prognosis-related
immune genes, and the correlations between CNV and
immune cell abundance, and between immune cells and
survival were further assessed. SIGMAR1 ADRB1, also called β-1 adrenergic receptor
(AR), is a member of the G-protein coupled receptor
family and an important target in various therapeutic
applications. In cardiomyocytes, proteinkinase A Figure 6. VEGFR1-specific signaling pathway that HIF1A
participates in. VEGFR1, vascular endothelial growth factor
receptor 1; HIF1A, hypoxia-inducible factor 1. We further conducted a series of in-depth analyses on
ADRB1. High amplification of ADRB1 in B cells and
dendritic cells might indicate that ADRB1 mutation can
facilitate two types of antigen presenting cells to
efficiently mediate and maintain a normal immune
response. The better prognosis in patients with high Figure 6. VEGFR1-specific signaling pathway that HIF1A
participates in. VEGFR1, vascular endothelial growth factor
receptor 1; HIF1A, hypoxia-inducible factor 1. AGING 359 www.aging-us.com Functional enrichment analysis The gene ontology categories including biological
processes,
molecular
functions,
and
cellular
components were assessed for DEGs. Moreover, to
determine whether ADRB1-related pathways were
statistically and consistently different between the high
and low TMB groups, we performed pathway
enrichment analysis using the GSEA software (version
4.0.3)
with
FDR<0.05
considered
statistically
significant. Analysis of mutation genes BRCA samples of TCGA were assessed using the R
package “BiocManager” MAF files containing somatic
variants and visualized with the maftools package. TMB
was obtained by calculating the number of tumor
mutations per Mb in each sample. The survival curve
was plotted to present the survival rate in relation to
TMB. A p-value < 0.05 was considered significant. The
limma package was performed to assess the relationship
between TMB and clinical characteristics including age,
sex, and the stages T, N, and M (p<0.05). Data collection Gene expression profiling for BRCA tissue samples
(n=109, t=1109) and patients’ clinical data (n=1097)
were
downloaded
from
the
TCGA
portal
(https://portal.gdc.cancer.gov/) (Data Release 24.0 -May
07, 2020). In addition, tumor mutation data (n=1044) of
BRCA including the names of the mutation genes, the
mutation types, and the mutation locations were
obtained from the “SomaticSniper variant aggregation
and masking” platform. Co-analyses of TMB and immune infiltration levels of B cells also supports this observation. In
addition to CNV, molecular research has demonstrated
that the transcription factor HIF1A drives tumor growth
and metastasis, and is associated with poor prognosis in
BRCA [37]. The inhibition of HIF1A pathway
activation combined with β-blockers may be a
promising treatment strategy for BRCA patients. In
addition, 17 small-molecule drugs targeting ADRB1
and other cancer-related genes obtained in this study
also support this proposed treatment. The deconvolution algorithm CIBERSORT [38] was
used to evaluate the relative abundance of immune cells
and the gene expression of tissue samples utilizing the
gene expression characterization system of 22 different
tumor-infiltrating lymphocyte subsets. The number of
permutations was set to 1000, and a p-value <0.05 was
regarded as successful. The immune cell matrix was
obtained for each sample in the transcriptome data by
the CIBERSORT R script v1.03. Similarly, the intrinsic
differences in the abundance of immune cells between
the high and low TMB groups were further explored
and visualized by bar plots. The differences of immune
cell infiltration between the high and low TMB groups
were visualized by violin plots. Additionally, the list of
immunologically relevant genes was downloaded from
the ImmPort database (https://www.immport.org/) (Data
Release 34, April 2020). Immune genes in DEGs were
screened out using the Venn diagram. The univariate
cox regression analysis was performed to identify
prognosis-related immune genes (p<0.05). In conclusion, our study identified prognosis-related
immune genes in BRCA mutations based on a co-analysis
of TMB and immune infiltration, and explored the intrinsic
correlation between TMB and immune infiltration. ADRB1 was identified as a potential biomarker for
BRCA, which may provide new insights for co-therapy. FUNDING This work is supported by the grants from National
Natural Science Foundation of China (81673799,
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Acute Aortic Dissection Presenting Exclusively as Lower Extremity Paresthesias
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UC Irvine
Journal of Education and Teaching in Emergency Medicine
Title
Acute Aortic Dissection Presenting Exclusively as Lower Extremity Paresthesias
Permalink
https://escholarship.org/uc/item/9b32n071
Journal
Journal of Education and Teaching in Emergency Medicine, 2(2)
Authors
Gibney, Ryan
Patane, Jonathan
Bunch, Steven
Publication Date
2017
DOI
10.5070/M522034559
Copyright Information
Copyright 2017 by the author(s).This work is made available under the terms of a Creative
Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/
Peer reviewed UC Irvine
Journal of Education and Teaching in Emergency Medicine
Title
Acute Aortic Dissection Presenting Exclusively as Lower Extremity Paresthesias
Permalink
https://escholarship.org/uc/item/9b32n071
Journal
Journal of Education and Teaching in Emergency Medicine, 2(2)
Authors
Gibney, Ryan
Patane, Jonathan
Bunch, Steven
Publication Date
2017
DOI
10.5070/M522034559
Copyright Information
Copyright 2017 by the author(s).This work is made available under the terms of a Creative
Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/
Peer reviewed UC Irvine
Journal of Education and Teaching in Emergency Medicine
Title
Acute Aortic Dissection Presenting Exclusively as Lower Extremity Paresthesias
Permalink
https://escholarship.org/uc/item/9b32n071
Journal
Journal of Education and Teaching in Emergency Medicine, 2(2)
Authors
Gibney, Ryan
Patane, Jonathan
Bunch, Steven
Publication Date
2017
DOI
10.5070/M522034559
Copyright Information
Copyright 2017 by the author(s).This work is made available under the terms of a Cre
Commons Attribution License, available at https://creativecommons.org/licenses/by/4
Peer reviewed Copyright Information
Copyright 2017 by the author(s).This work is made available under the terms of a Creative
Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Powered by the California Digital Library
University of California eScholarship.org ney, BS , Jonathan Patane, MD and Steven Bunch, MD
of California, Irvine, Department of Emergency Medicine, Orange, CA
should be addressed to Ryan Gibney, BS at rgibney@uci.edu
ary 9, 2017; Accepted: March 22, 2017; Electronically Published: April 15,2016; https://doi.org/10.21980/J8NK57
7 Gibney, et al. This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) License. S
mmons.org/licenses/by/4.0/
. 3 3 Video Link: https://youtu.be/4VPCQHDOTg4
https://youtu.be/yBBvSRrqDJw Video Link: https://youtu.be/4VPCQHDOTg4
https://youtu.be/yBBvSRrqDJw Video Link: https://youtu.be/4VPCQHDOTg4
https://youtu.be/yBBvSRrqDJw History of present illness: A 45-year-old male presented with left-lower extremity numbness for 3 hours. He denied chest pain, shortness of breath, back pain, or pain to his leg. On physical examination, he was
noted to have a normal cardiac exam without murmurs and normal breath sounds, but had no palpable
femoral or dorsalis pedis pulse on the left lower extremity. His extremity examination showed normal range
of motion, full strength to both lower extremities, but subjective decreased sensation over the entire left
lower extremity in all dermatomes. Significant findings: Chest X-ray and computed tomography (CT) angiogram was performed to evaluate his
thoracic and abdominal vasculature. Chest X-ray did not show any significant widening of the mediastinum. The CT angiogram demonstrated an intimal tear along the aortic arch separating a true and false aortic
lumen, consistent with an acute aortic dissection. The true lumen (highlighted in blue in images 1-5) can be
identified by continuity with an undissected part of the aorta.1 While the false lumen (highlighted in red in
images 1-5) can be identified by its crescent shape and larger cross-sectional area.1 Discussion: The classic acute aortic dissection patient presents with sudden onset, tearing chest pain
radiating to the back. Inconsistent blood pressure in the extremities is often found, along with diminished or
absent peripheral pulses and widened mediastinum on chest X-ray. Painless dissection rarely occurs (6.4% of
cases), and is more often found in older patients and is associated with higher mortality.2 Although rare,
isolated lower limb findings such as ischemia or paresthesias are noted to occur in about 10% of cases.2 When
aortic dissection presents solely with extremity complaints, the diagnosis is often missed.3 Computed 4 4 4 tomography angiography is the ideal imaging modality to confirm the diagnosis and classification. Imaging
can be obtained in stable patients in conjunction with emergent surgical consultation, pain control, and
decreasing the heart rate and blood pressure. Esmolol is considered the first line agent for decreased both
heart rate and blood pressure, simultaneously, to reduce the shear stress of the wall of the dissecting
vasculature.4 Topics: Acute aortic dissection, distal limb ischemia. 5 References: References:
1. Castañer E, Andreu M, Gallardo X, Mata JM, Cabezuelo MA, Pallardó Y. CT in nontraumatic acute thoracic aortic disease:
typical and atypical features and complications. Radiographics. 2003; 23: S93-110. doi: 10.1148/rg.23si035507
2. Park S, Hutchinson S, Mehta R, Isselbacher E, Cooper JV, Fang J, et. al. Association of painless acute aortic dissection with
increased mortality. Mayo Clin Proc. 2004; 79:125-1257. doi: 10.4065/79.10.1252
3. Lee C, Chang C, Tsai Y, Wu C. Isolated lower limb ischemia as an unusual presenting symptom of aortic dissection. Cardovasc 1. Castañer E, Andreu M, Gallardo X, Mata JM, Cabezuelo MA, Pallardó Y. CT in nontraumatic acute thoracic aortic disease:
typical and atypical features and complications. Radiographics. 2003; 23: S93-110. doi: 10.1148/rg.23si035507 2. Park S, Hutchinson S, Mehta R, Isselbacher E, Cooper JV, Fang J, et. al. Association of painless acute aortic dissection with
increased mortality. Mayo Clin Proc. 2004; 79:125-1257. doi: 10.4065/79.10.1252 3. Lee C, Chang C, Tsai Y, Wu C. Isolated lower limb ischemia as an unusual presenting symptom of aortic dissection. Cardovasc
J Afr. 2012;23:13-14. 4. Hiratzka LF, Bakris GL, Beckman JA, et al. 2010 ACCF/AHA/AATS/ACR/ASA/SCA/SCAI/SIR/STS/SVM guidelines for the
diagnosis and management of patients with thoracic aortic disease: a report of the American College of Cardiology
Foundation/American Heart Association Task Force on Practice Guidelines, American Association for Thoracic Surgery,
American College of Radiology, American Stroke Association, Society of Cardiovascular Anesthesiologists, Society for
Cardiovascular Angiography and Interventions, Society of Interventional Radiology, Society of Thoracic Surgeons, and Society
for Vascular Medicine. Circulation. 2010;121:e266. doi: 10.1161/CIR.0b013e3181d4739e 4. Hiratzka LF, Bakris GL, Beckman JA, et al. 2010 ACCF/AHA/AATS/ACR/ASA/SCA/SCAI/SIR/STS/SVM guidelines for the
diagnosis and management of patients with thoracic aortic disease: a report of the American College of Cardiology
Foundation/American Heart Association Task Force on Practice Guidelines, American Association for Thoracic Surgery,
American College of Radiology, American Stroke Association, Society of Cardiovascular Anesthesiologists, Society for
Cardiovascular Angiography and Interventions, Society of Interventional Radiology, Society of Thoracic Surgeons, and Society
for Vascular Medicine. Circulation. 2010;121:e266. doi: 10.1161/CIR.0b013e3181d4739e 5 5
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Mediating Effect of Self-management on Health Empowerment and Quality of Life of Elderly Comorbid Patients
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Research Square (Research Square)
| 2,023
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cc-by
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Mediating Effect of Self-management on Health
Empowerment and Quality of Life of Elderly
Comorbid Patients Lanxin Wu
Zhengzhou University
Yan Zhang
(
zhangyanmy@126.com
)
Zhengzhou University
Lixue Meng
Zhengzhou University
Li Liu
Zhengzhou University
Ting Zhao
Zhengzhou University Objective To explore the relationship among health empowerment, self-management and quality of life of elderly
patients with comorbidity. Results The score of health empowerment, self-management and quality of life of the elderly patients with
comorbidity were (92.18±8.917), (136.58±12.42) and (89.30±8.72) respectively. Health empowerment,
self-management and quality of life of elderly patients with comorbidity were positively correlated (P <
0.01). The direct effect of health empowerment on quality of life is 0.493, the indirect effect is 0.207, the
total effect is 0.724, and the intermediary effect accounts for 28.75% of the total effect. Self-
management plays a partial intermediary role between health empowerment and quality of life. Research Article Keywords: senile comorbidity, Self-management, Health empowerment, Quality of life, mesomeric effect
Posted Date: May 17th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2898513/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Posted Date: May 17th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2898513/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Page 1/13 Page 1/13 Conclusion The health empowerment, self-management and quality of life of elderly comorbid patients need to be
further improved. The self-management of elderly comorbid patients plays a partial intermediary role
between health empowerment and quality of life, and the quality of life of patients can be effectively
improved by improving their health empowerment and self-management ability. Background Under the general trend of global aging, comorbidity among the elderly is becoming more and more
common, which may have a certain impact on the quality of life of the elder. There is evidence that self-
management and health empowerment are positively correlated with quality of life. However, limited
research has explored the potential mechanism among them. Therefore, the purpose of this study is to
explore the interaction path and mechanism among health empowerment, self-management and quality
of life of elderly comorbid patients, and to provide theoretical basis for formulating intervention programs
to improve their quality of life in the later stage. Methods 309 elderly patients with comorbidity in Zhengzhou City, Henan Province were investigated by
questionnaire, including Health Empowerment Scale, Chronic Disease Self-management Scale and SF-12
Scale. Introduction According to the data of the seventh national census of the National Bureau of Statistics, the population
aged 60 and above is 260 million, accounting for 18.70%[1]. It is estimated that by 2050, the elderly Page 2/13 population aged 60 and above in China will be close to 500 million, accounting for over one third of the
total population [2]. With the increase of life expectancy, the number of elderly people with chronic
diseases increases. About 15.3–91.3% of the global population suffers from comorbidity [3]. According
to the latest data of British Medical Journal (BMJ), the phenomenon of comorbidity in high-income
countries in the world is mainly driven by age. With the change of population structure, the proportion of
people suffering from two or more diseases is increasing steadily [4]. The Report on Nutrition and Chronic
Diseases of Residents in China (2020) pointed out that the aging degree of the population in China is
deepening, and the incidence of various chronic diseases, mainly hypertension, diabetes and chronic
obstructive pulmonary disease, is rising, especially the comorbidity of the elderly [5]. Comorbidity not only
reduces the functional status and quality of life of the elderly, but also significantly increases the risk of
disability, weakness, death and multiple drug use, resulting in an increasing demand for medical security,
which puts forward higher requirements for the health management of comorbidity. Self-management refers to a series of health actions taken by patients to cope with diseases, reduce the
occurrence of complications and alleviate the negative effects of diseases on their social activities and
psychological factors [6].Self-management can effectively improve patients' compliance, subjective
initiative and quality of life [7]. In China, the self-management of elderly patients with chronic diseases is
still at a low level [8]. Most elderly people have limited knowledge of diseases, and their poor
management may lead to additional complications and medical expenses, and at the same time have a
certain impact on their quality of life [9]. Studies have shown that self-management of chronic diseases
can improve the health status and quality of life of elderly patients with chronic diseases and reduce the
incidence of complications [10]. Introduction Health empowerment is a positive self-care strategy and cooperative relationship, which can promote the
elderly to gradually manage themselves and empower others by stimulating their internal responsibility
and gaining external social support, so as to achieve the goal of improving health outcomes and quality
of life [11]. Studies have shown that improving the health empowerment ability of elderly patients with
comorbidity can effectively improve their disease management ability, and then improve the quality of life
of patients [12]. At present, there are many studies on the single predictive function of self-management,
health empowerment and quality of life, and there are few studies on the comprehensive investigation of
the three mechanisms. This study will investigate the interaction path and mechanism among health
empowerment, self-management and quality of life of elderly comorbid patients, and provide theoretical
basis for formulating intervention programs to improve their quality of life in the later stage. General information questionnaire The researcher designed it by himself after consulting the literature, including: age, gender, nationality,
residence, education level, marital status, religious belief, monthly income, occupation, residence, medical
payment method and chronic diseases. Chronic disease self-management scale In this study, Liu Hongxia [13] revised the Chronic Disease Self-management Scale. It is mainly used to
measure the cognitive ability, psychological quality, lifestyle and treatment compliance of the subjects. The scale consists of 4 first-level indicators and 40 second-level indicators. It was divided into five grades
of "none, few, sometimes, often and always" by Likert5-level scoring method, and each grade is given "1
point, 2 points, 3 points, 4 points and 5 points" respectively. The Cronbach'α coefficient of the scale is
0.919, and the reliability is good. Health empowerment scale Measurement of the health empowerment scale for chronic diseases in the elderly developed by Yang
Yang [14]. It includes five dimensions: responsibility belief, obtaining support, increasing knowledge,
participating in treatment and rebuilding oneself, with a total of 26 items. 1 is "very different" and 5 is
"very agree". The higher the score, the higher the awareness of health empowerment. Cronbach'α = 0.927,
which shows that the scale has good reliability. The average score of this study is > 3 for high level, 3 for
medium level and < 3 for low level. Study design and participants This cross-sectional study was conducted from January to March, 2023. The elderly comorbid patients in
Jinshui District, Erqi District and Zhongyuan District of Zhengzhou City, Henan Province were selected by
cluster sampling method as the research objects. Inclusion criteria: ① Age ≥ 60 years old; (2) Meet the Page 3/13 Page 3/13 WHO diagnostic criteria of hypertension, coronary heart disease, cerebral infarction, diabetes, chronic
obstructive pulmonary disease (COPD) and osteoarthritis; Determine the types of diseases with reference
to the health management files of community residents in community health service centers; ③ Those
with normal reading, understanding and expression can be assessed for their physical ability; ④ Informed
consent. Exclusion criteria: ① severe mental disorder; ② Those who are participating in other clinical trials. Before data collection, researchers will explain the purpose of the study to patients and obtain the
informed consent of each patient. Investigators used questionnaires to conduct face-to-face surveys. After eliminating the invalid questionnaires, 309 valid questionnaires were finally included in the
analysis.All older adults gave full informed written consent for participation.The Ethics Committee of
Zhengzhou University approved the study.The study conforms to the declaration of Helsinki. SF-12 scale Page 4/13 SF-12 is a 12-item short-form health survey (SF-12) in the United States. It is a simplified version of the
36-item short-form health survey (SF-12) in the United States, which is widely used in the world at present. It is a universal scale to measure life in the past four weeks. Compared with SF-36, it has the advantages
of simple entry and less operation time. There are 12 items with 8 dimensions, namely, general health
(GH), physical function in (PF), role-physical,Rp function (RP), body pain (BP), vitality(VT), social
functioning(SF), role-emotional(RE),function(RE), mental health (MH). Each item is graded according to
the corresponding options, among which questions 1, 8, 9 and 10 are graded in reverse. SF-12 can
calculate physcial component summary and mental component summary(MCS). The higher the score,
the better the quality of life. General information In this study, 309 data were distributed and 309 data were recovered, and the effective recovery rate was
100%, including 118 males (38.2%) and 191 females (61.8%). There were 55 cases aged 60–69 years,
accounting for 17.8%, 132 cases aged 70–79 years, accounting for 42.7%, and 122 cases ≥ 80 years,
accounting for 39.5%. There are 135 cases (43.7%) of primary school or below, 94 cases (30.4%) of junior
high school, 39 cases (12.6%) of senior high school, and 41 cases (13.3%) of junior college or above. See
Table 1 for details. Total score and dimensions of health empowerment, self-management and quality of life of elderly
patients with comorbidity The score of quality of life of elderly patients with comorbidity was (89.30 ± 8.72), including physical
health (38.19 ± 7.25), mental health (51.05 ± 5.33) and self-management (136.58 ± 12.42), among which
the score of cognitive ability dimension (37.28 ± 6.29). The scores of lifestyle dimension, treatment
compliance dimension and health empowerment were (51.55 ± 4.49), (26.40 ± 3.76) and (92.18 ± 8.917)
respectively. Statistical analysis Statistical analysis was carried out by SPSS25.0 software, the measurement data were expressed by
mean standard deviation, and the correlation analysis data conformed to normal distribution by Pearson
correlation analysis. Stepwise linear regression was used to test the mediating effect of self-management
on health empowerment and quality of life of elderly patients with comorbidity. P < 0.05 was statistically
significant. Mediating Effect of Self-management on Health Empowerment and
Quality of Life of Elderly Comorbid Patients Control the influence of demographic variables, and verify the mediation effect of self-management
between health empowerment and quality of life by using the mediation effect test procedure. The first
step is to make regression analysis with health empowerment as independent variable and quality of life
as dependent variable. The results show that health empowerment can positively predict quality of life (β
= 0.443, P < 0.001). The second step is to make a regression analysis with health empowerment as the
independent variable and self-management as the dependent variable. The results show that health
empowerment can positively predict self-management ability (β = 0.547, P < 0.001). The third step is to
make regression analysis with health empowerment and self-management as independent variables and
quality of life as dependent variables. The results show that health empowerment and self-management
can positively predict quality of life (β = 0.443, P < 0.01). Self-management plays a significant mediating
role between health empowerment and quality of life of elderly patients with comorbidity, as shown in
Table 3. Correlation analysis of health empowerment, self-management and
quality of life of elderly patients with comorbidity Page 5/13 The results of correlation analysis show that there are correlations among self-management, health
empowerment and quality of life of elderly patients with comorbidity, which are statistically significant (P
< 0.01). See Table 2 for details. Set the initial model: health empowerment has direct and indirect effects on the quality of life, and self-
management plays an intermediary role. The direct effect of health empowerment on quality of life is 0.493, the mediating effect is 0.547×0.379 =
0.207, the total effect is 0.519 + 0.207 = 0.724, and the mediating effect accounts for
0.207/0.724×100%=28.75% of the total effect. Therefore, self-management plays a partial intermediary
role between health empowerment and quality of life, as shown in Fig. 1. Health empowerment, self-management and quality of life of elderly patients with comorbidity are
positively correlated. Health empowerment, self-management and quality of life of elderly patients with comorbidity are
positively correlated. The results of this study show that there is a positive correlation between health empowerment and
quality of life (r = 0.493, P < 0.01). That is, the higher the health empowerment, the better the quality of life
of patients. Health empowerment is the process and result that individual gain knowledge and ability,
strengthen their beliefs, change their healthy behaviors, enhance their sense of self-awareness, and gain
self-development and self-satisfaction, thus controlling diseases and promoting health [20]. Many studies
show that [21–23], health empowerment can trigger behavioral changes of patients with chronic
diseases, and increase their health knowledge, self-management, quality of life and disease resilience. The results of this study show that self-management is positively correlated with quality of life (r = 0.379,
P = 0.017). That is, the higher the self-management ability, the better the quality of life. Research shows
that self-management can reduce patients' pain (depression and anxiety) and self-denial emotions,
effectively improve patients' compliance, enhance their subjective initiative and improve their quality of
life [24]. Based on the above, medical staff should fully realize that a higher level of health empowerment
can effectively promote the elderly to improve their disease awareness and self-management behavior
ability and obtain a higher quality of life. In the future research, we should constantly tap the internal and
external resources of health empowerment, improve its self-management level, and then improve the
quality of life. The health empowerment, self-management and quality of life of elderly patients with comorbidity need
to be improved. The health empowerment, self-management and quality of life of elderly patients with comorbidity need
to be improved. This study shows that the health empowerment, self-management and quality of life of elderly patients
with comorbidity still need to be improved. Qian Yan [15], Yang Yang [16], Yu Jun [6] and others have
higher scores in self-management, health empowerment and quality of life of elderly patients with
diabetes, elderly patients with chronic diseases, and middle-aged and elderly patients with AIDS than this
study. Perhaps the above subjects are patients with a single disease, and this study is patients with
multiple diseases, which increases the difficulty of disease management and greatly reduces the
confidence and ability of patients in disease management. Many studies show that [17, 18], the ability of
health empowerment and self-management can improve the quality of life of patients with chronic Page 6/13 diseases. As a self-care strategy to improve health outcomes and quality of life, health empowerment can
improve the self-management ability of the elderly and play a key role in promoting and improving health
[19]. Therefore, simple and effective intervention measures need to be taken to establish the self-
management ability and health empowerment ability of elderly patients with comorbidity, so as to
improve their quality of life. Funding The study was financially supported by the National Natural Science Foun The study was financially supported by the National Natural Science Foundation of China. nancially supported by the National Natural Science Foundation of China. Self-management plays a partial intermediary role between health empowerment and quality of life of
elderly patients with comorbidity. Mediating effect analysis shows that self-management plays a partial mediating role between health
empowerment and quality of life, accounting for 28.75% of the total effect, and the mediating effect of
self-management between health empowerment and quality of life is 0.483. This shows that health
empowerment can not only directly affect the quality of life, but also indirectly affect the quality of life
through self-management. It may be that the greater patients' confidence in disease management, the
stronger their sense of responsibility for their own disease management, and the more they tend to
actively seek health-related information, which is more conducive to their self-management, reducing the
impact of diseases on their lives and improving their quality of life. Therefore, when considering how to
improve the quality of life of elderly comorbid patients, medical staff should not only pay attention to
improving patients' health empowerment ability, but also pay attention to patients' self-management
ability, and establish a perfect and comprehensive disease management model through various channels
and forms. In addition, when making a plan, we should comprehensively consider the patient's personal Page 7/13 Page 7/13 situation and make a personalized management plan, so that patients can have good self-management
ability in different environments and improve their quality of life. situation and make a personalized management plan, so that patients can have good self-management
ability in different environments and improve their quality of life. Limitation There are some limitations in this study: Firstly, it is only a cross-sectional study, and it can be extended
to a longitudinal study to explore its variable relationship in the future; Secondly, Future research can
expand the sample size and explore the influence of multiple factors on patients' health outcomes. Conclusion self-management, health empowerment and quality of life of elderly patients with comorbidity still need
to be improved. Self-management and health empowerment are positively related to quality of life, and
self-management has some mediating effects between health empowerment and quality of life. Therefore, in the future research, we can learn from the mechanism among them to formulate
intervention programs for elderly patients with comorbidity, improve their self-management and health
empowerment, and then improve their quality of life and health outcomes. Acknowledgments The authors thank the participants for their support to this study. Authors’ contributions Wu Lanxin and Zhang Yan designed the study.Wu Lanxin and Liu Li distributed the questionnaire, Wu
Lanxin analyzed the data.Zhang Yan revised the article, all authors read and participated in revising the
article and approved its submission to BMC Public Health Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available due to the
data containing information that could compromise research participant privacy/consent, but are
available from the researcher Wu Lanxin (1094667097@qq.com) on reasonable request, and subject to
approval from the research committee of the Zhengzhou University. Ethics approval and consent to participate Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional
affiliations. Ethics approval and consent to participate All methods were carried out in accordance with relevant guidelines and regulations.This study was
conducted with the approval from the Ethics Committee of Zhengzhou University, (ZZUIRB2023-069). All
participants included in the study provided informed consent.The aim and scope of the research were
explained at the beginning of the survey in the questionnaire. A sentence on voluntary informed consent
was added at the beginning of the questionnaire and participants that did not give voluntary informed
consent were not allowed to continue the survey. Consent for publication Not Applicable. Competing interests The authors declare no competing interests. References PMID: 36447176; PMCID: PMC9710041. 10. Kim S, Park M, Song R. Effects of self-management programs on behavioral modification among
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review[J]. BMC Infectious Diseases, 2021,21(1). Tables Tables
Table 1 General data of elderly comorbid patients Table 1 General data of elderly comorbid patients Page 11/13 Table 2 Correlations of health empowerment, self-management, and quality of life in elderly patients with
comorbidities
Table 3 Regression analysis of the variable relationships in the mediation model Table 2 Correlations of health empowerment, self-management, and quality of life in elderly patients with
comorbidities Table 2 Correlations of health empowerment, self-management, and quality of life in elderly patients with
comorbidities Table 3 Regression analysis of the variable relationships in the mediation model Table 3 Regression analysis of the variable relationships in the mediation model Regression analysis of the variable relationships in the mediation model Figures Page 12/13
Figure 1 Figure 1 Mediation effect model of health empowerment in self-management and quality of life in elderly patients
with comorbidities Mediation effect model of health empowerment in self-management and quality of life in elderly patients
with comorbidities Page 13/13
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Apolipoprotein E gene polymorphism: effects on plasma lipids and risk of type 2 diabetes and coronary artery disease
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Cardiovascular diabetology
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* Correspondence: atip.lik@mahidol.ac.th
1Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol
University, Bangkok 10700, Thailand
Full list of author information is available at the end of the article ORIGINAL INVESTIGATION Open Access Abstract Background: The most common apolipoprotein E (apoE) gene polymorphism has been found to influence plasma
lipid concentration and its correlation with coronary artery disease (CAD) has been extensively investigated in the
last decade. It is, however, unclear whether apoE gene polymorphism is also associated with increased risk of type
2 diabetes mellitus (T2DM). The knowledge of this study may provide the primary prevention for T2DM and CAD
development before its initiation and progression. Therefore, this study was carried out to determine the
association between apoE gene polymorphism and T2DM with and without CAD and its role in lipid metabolism. Methods: The case-control study was carried out on a total of 451 samples including 149 normal control subjects,
155 subjects with T2DM, and 147 subjects with T2DM complicated with CAD. The apoE gene polymorphism was
tested by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Univariable and
multivariable logistic regression analyses were used to identify the possible risks of T2DM and CAD. Results: A significantly increased frequency of E3/E4 genotype was observed only in T2DM with CAD group (p =
0.0004), whereas the ε4 allele was significantly higher in both T2DM (p = 0.047) and T2DM with CAD (p = 0.009) as
compared with controls. E3/E4 genotype was also the independent risk in developing CAD after adjusting with
established risk factors with adjusted odds ratio (OR) 2.52 (95%CI 1.28-4.97, p = 0.008). The independent predictor
of individuals carrying ε4 allele still remained significantly associated with both CAD (adjusted OR 2.32, 95%CI 1.17-
4.61, p = 0.016) and T2DM (adjusted OR 2.04, 95%CI 1.07-3.86, p = 0.029). After simultaneously examining the joint
association of E3/E4 genotype combined with either obesity or smoking the risk increased to approximately 5-fold
in T2DM (adjusted OR 4.93, 95%CI 1.74-13.98, p = 0.003) and 10-fold in CAD (adjusted OR 10.48, 95%CI 3.56-30.79,
p < 0.0001). The association between apoE genotypes on plasma lipid levels was compared between E3/E3 as a
reference and E4-bearing genotypes. E4-bearing genotypes showed lower HDL-C and higher VLDL-C and TG,
whereas other values of plasma lipid concentrations showed no significant difference. Conclusions: These results indicate that ε4 allele has influence on lipid profiles and is associated with the
development of both T2DM with and without CAD, and furthermore, it increased the risk among the subjects with
obesity and/or smoking, the conditions associated with high oxidative stress. CARDIO
VASCULAR
DIABETOLOGY CARDIO
VASCULAR
DIABETOLOGY Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 CARDIO
VASCULAR
DIABETOLOGY © 2012 Chaudhary et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Apolipoprotein E gene polymorphism: effects on
plasma lipids and risk of type 2 diabetes and
coronary artery disease Rajesh Chaudhary1, Atip Likidlilid1*, Thavatchai Peerapatdit2, Damras Tresukosol2, Sorachai Srisuma3,
Suphachai Ratanamaneechat4 and Charn Sriratanasathavorn5 * Correspondence: atip.lik@mahidol.ac.th
1Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol
University, Bangkok 10700, Thailand
Full list of author information is available at the end of the article
© 2012 Chaudhary et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Introduction The prevalence of T2DM and CAD is increasing in
Thai population according to the cross-sectional ECG
survey of 1991 in Thai population, which found that the
age-standardized prevalence rate of CAD was 9.9/1000
subjects (men 9.2/1000, women 10.7/1000) [19]. World
Health Organization (WHO) global prevalence of dia-
betes report estimated that by the year 2030, 366 million
people, particularly in developing countries, will be
affected by diabetes [20]. Genetic factors like apoE are Type 2 diabetes mellitus (T2DM) is one of the most com-
mon diseases with a high incidence and prevalence
throughout the world. It affects nearly 4% of the world’s
population and this percentage will supposedly be increas-
ing up to 5.4% by year 2025 [1]. Prevalence of diabetes in
Thai adults as shown by the previous study on Thai popu-
lation was 9.6% (2.4 million population) and the impaired
fasting glucose was 5.4% (1.4 million people). Mean fasting
plasma glucose level by age, sex, and area of residence was
found to be substantially higher in urban population
group than rural [2]. T2DM is also known as a major
independent risk factor for coronary artery disease (CAD)
and is the major cause of morbidity and mortality affecting
people with diabetes. To date, several mechanisms such as
dyslipoproteinemia, obesity, oxidative stress, smoking,
exercise, alcohol intake, and genetic factors have been
identified as risk factors of both T2DM and CAD. Lack of
apolipoprotein E (apoE) gene has been clearly demon-
strated as a leading cause of severe hyperlipidemia and
spontaneous development of atherosclerosis in mammals
[3,4]. However, few studies have been able to demonstrate
an association between T2DM and various single nucleo-
tide polymorphisms (SNPs) [5]. Recently, Zeljko et al. 2011 [6] indicated that apoE gene polymorphism is also
associated with obesity in normal Croatian Roma popula-
tion. Adipocytes in an obese person which are the central
and causal components in T2DM can generate high
amount of biologically active molecules called adipokines
or adipocytokines such as plasminogen activator inhibitor-
1 (PAI-1), resistin, leptin, interleukin-6 (IL-6), and tumor
necrosis factor alpha (TNF-a) [7]. These inflammatory
cytokines inhibit insulin-stimulated glucose metabolism in
skeletal muscles and stimulate gluconeogenesis in hepato-
cytes causing hyperglycemia [8]. Hyperglycemia-induced
oxidative stress results in reducing glucose uptake from
blood by muscle cells and develops into insulin resistance
by decreasing insulin secretion from pancreatic b-cells [9]. Abstract Consequently, with their ability to affect
lipid levels, the apoE gene polymorphism could be one of
the factors influencing development of both T2DM and
CAD. The prevalence of T2DM and CAD is increasing in
Thai population according to the cross-sectional ECG
survey of 1991 in Thai population, which found that the
age-standardized prevalence rate of CAD was 9.9/1000
subjects (men 9.2/1000, women 10.7/1000) [19]. World
Health Organization (WHO) global prevalence of dia- Abstract Keywords: Apolipoprotein E, Polymorphism, Type 2 diabetes mellitus, Hyperglycemia, Coronary artery disease,
Restriction fragment length polymorphism Page 2 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 lipoproteins in our body and also responsible for the
development of CAD [10,11]. ApoE acts as a high affinity
ligand for several hepatic lipoprotein receptors such as
low-density lipoprotein receptor (LDLR) and LDL-related
protein (LRP) and is involved in the process of cellular
incorporation of several lipoproteins for transport and
digestion [12]. ApoE is a plasma glycoprotein of 34 kDa
with 299-amino acids associated with several other
plasma glycoproteins, such as high density lipoprotein
(HDL), very low density lipoprotein (VLDL), and chylo-
microns [13]. In humans, apoE gene located on the chro-
mosome at position 19q13.2 has been known to be
polymorphic. SNPs at positions 112 (rs 429358) and 158
(rs 7412) determine three major alleles: ε2 (T to C substi-
tution at position 158), the most common ε3, and ε4 (C
to T substitution at position 112); 3 isoforms: ApoE2
(Cys112, 158Cys), ApoE3 (Cys112, 158Arg), and ApoE4
(Arg112, 158Arg); and 6 genotypes having 3 homozygous:
E2/E2, E3/E3, E4/E4, and 3 heterozygous: E2/E3, E2/E4,
E3/E4 [13]. Previous studies have shown that apoE alleles
have influence on the lipid clearance and metabolism in
humans. ApoE ε2 allele has been reported to be asso-
ciated with higher plasma levels of apoE, decreased
plasma levels of LDL cholesterol (LDL-C) and lower risk
of CAD [14] while apoE ε4 is associated with lower
plasma level of apoE, increased plasma levels of total cho-
lesterol (TC), LDL-C, VLDL cholesterol (VLDL-C), and
greater risk of CAD when compared to apoE3 homozy-
gotes [15]. One reason for this impaired clearance by
apoE ε4 leading to pathogenesis of CAD might be that
apoE ε4 binds strongly to LDLR compared to other geno-
types. The resulting high amount of lipid can suppress
the synthesis of LDLR leading to lower clearance of lipo-
protein from our body through LDLR [15]. Other studies
have also supported that apoE ε4 allele is associated with
the risk of CAD [16]. However, according to a recent
meta-analysis, the cardiovascular role of apoE2 is uncer-
tain [17] because of its tendency to increase triglyceride
(TG) level [18]. In addition, apo ε2 homozygote in com-
bination with certain additional disorders may develop
type III familial hyperlipidemia and premature athero-
sclerosis [12]. Introduction lipoproteins in our body and also responsible for the
development of CAD [10,11]. ApoE acts as a high affinity
ligand for several hepatic lipoprotein receptors such as
low-density lipoprotein receptor (LDLR) and LDL-related
protein (LRP) and is involved in the process of cellular
incorporation of several lipoproteins for transport and
digestion [12]. ApoE is a plasma glycoprotein of 34 kDa
with 299-amino acids associated with several other
plasma glycoproteins, such as high density lipoprotein
(HDL), very low density lipoprotein (VLDL), and chylo-
microns [13]. In humans, apoE gene located on the chro-
mosome at position 19q13.2 has been known to be
polymorphic. SNPs at positions 112 (rs 429358) and 158
(rs 7412) determine three major alleles: ε2 (T to C substi-
tution at position 158), the most common ε3, and ε4 (C
to T substitution at position 112); 3 isoforms: ApoE2
(Cys112, 158Cys), ApoE3 (Cys112, 158Arg), and ApoE4
(Arg112, 158Arg); and 6 genotypes having 3 homozygous:
E2/E2, E3/E3, E4/E4, and 3 heterozygous: E2/E3, E2/E4,
E3/E4 [13]. Previous studies have shown that apoE alleles
have influence on the lipid clearance and metabolism in
humans. ApoE ε2 allele has been reported to be asso-
ciated with higher plasma levels of apoE, decreased
plasma levels of LDL cholesterol (LDL-C) and lower risk
of CAD [14] while apoE ε4 is associated with lower
plasma level of apoE, increased plasma levels of total cho-
lesterol (TC), LDL-C, VLDL cholesterol (VLDL-C), and
greater risk of CAD when compared to apoE3 homozy-
gotes [15]. One reason for this impaired clearance by
apoE ε4 leading to pathogenesis of CAD might be that
apoE ε4 binds strongly to LDLR compared to other geno-
types. The resulting high amount of lipid can suppress
the synthesis of LDLR leading to lower clearance of lipo-
protein from our body through LDLR [15]. Other studies
have also supported that apoE ε4 allele is associated with
the risk of CAD [16]. However, according to a recent
meta-analysis, the cardiovascular role of apoE2 is uncer-
tain [17] because of its tendency to increase triglyceride
(TG) level [18]. In addition, apo ε2 homozygote in com-
bination with certain additional disorders may develop
type III familial hyperlipidemia and premature athero-
sclerosis [12]. Consequently, with their ability to affect
lipid levels, the apoE gene polymorphism could be one of
the factors influencing development of both T2DM and
CAD. Materials and methods
Subjects T2DM complicated with CAD (T2DM + CAD) subjects
included 147 subjects. Subjects were confirmed CAD by
coronary angiography, with at least 50% stenosis in a
major coronary artery or one of their branches. Subjects
were diagnosed to have diabetes with FPG≥126 mg/dL or
those with drug-treated T2DM. Exclusion criteria included
those having renal disease, hepatic disease, type 1 diabetes
mellitus, any form of endocrine disease or metabolic
disease. The studied subjects were recruited from Siriraj Hospital
in Bangkok province. Full consent forms were signed by
the subjects after the nature and motif of the study was
clearly explained to them. The study protocol was
approved by the Ethics Committee of Clinical Study in
Humans, Faculty of Medicine Siriraj Hospital, Mahidol
University. Questionnaires were used to collect the infor-
mation of family and medical history, alcohol consump-
tion, smoking habits and physical activity. Other clinical
and biochemical data such as dyslipidemia, systolic blood
pressure (SBP) and diastolic blood pressure (DBP) from all
subjects were obtained from clinical and laboratory exami-
nations. Anthropometric data (weight, height) were col-
lected and used for BMI calculation. Obesity was defined
as BMI ≥25 kg/m2 according to WHO suggested criteria
for Asian populations [21]. Dyslipidemic or hyperlipidemic
were defined as when one has level of TC >200 mg/dL,
TG >150 mg/dL, LDL-C >130 mg/dL, HDL-C <40 mg/dL,
TC/HDL-C ratio >4.0 or under medication of lipid lower-
ing drugs. Cigarette smokers were allowed into the study
if they had once smoked even if they were no longer smo-
kers. Alcohol drinkers were defined as those who drank at
least two times a week for more than a year. Physical activ-
ities were defined as exercise for at least 2 to 3 days/week
for at least 30 minutes. Hypertension was defined as blood
pressure above 140/90 mmHg or taking antihypertensive
drugs. These subjects were categorized into three groups:
normal healthy controls, T2DM with and T2DM without
CAD, according to the criteria of American Diabetes
Association Classification 2010 [22], with an age range
from 40-65 years. APOE Genotyping Blood samples were collected in EDTA containing tubes. Guanidine-HCl precipitation method was performed for
genomic DNA extraction. Genomic DNA was subjected to
polymerase chain reaction (PCR) with primers specific to
apoE gene, sense: 5’ AACAACTGACCCCGGTGGCG 3’,
antisense: 5’ ATGGCGCTGAGGCCGCGCTC 3’, sense: 5’
CCCACCTGCGCAAGCTGCGC 3’, using thermal cycler
with thermal profile according to Richard et al [23]. In
brief, PCR reaction mixture included 20 pmol of each pri-
mer, 0.3 μg genomic DNA, 10 mM of each dNTP, 10X
PCR buffer, 10% DMSO in a final volume of 50 μL. 10 μL
of PCR products was digested with 0.3 unit of Hha1
enzyme according to the supplier’s recommended proce-
dure (Biolabs New England). The resulted fragments were
then separated on 8% polyacrylamide gel and stained with
ethidium bromide. Bands were compared with 25 bp DNA
marker and the different individual genotypes were sepa-
rated and categorized based on the following band length
criteria: E2/E2: 91, 83, 61; E3/E3: 91, 61, 48, 35; E4/E4: 72,
61, 48, 35; E2/E3: 91, 83, 61, 48, 35; E2/E4: 91, 83, 72, 61,
48, 35 and E3/E4: 91, 72, 61, 48, 35. Introduction Taken these together, increased oxidative stress in hyper-
glycemia and reduced lipid clearance because of apoE
gene polymorphism are effective in developing insulin
resistance and T2DM. In addition, high oxidative stress
can also cause vascular inflammation leading to athero-
sclerosis through several cytokines such as NF-B, TNF-a,
IL-1b, and other proinflammatory cytokines [10]. All of
these factors are one of the underlying causes of metabolic
syndrome [7]. Grundy reported that subjects suffering
from the metabolic syndrome are at 2-fold higher risk of
developing CAD and at 5-fold higher risk of developing
T2DM; however, persons suffering from T2DM are at
3-fold higher risk of developing CAD [11]. ApoE gene is one of the most studied genes which is
responsible for stabilizing and solubilizing circulating ApoE gene is one of the most studied genes which is
responsible for stabilizing and solubilizing circulating Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Page 3 of 11 T2DM subjects without CAD included 155 subjects. All subjects fulfilled the diabetes mellitus diagnostic cri-
teria of FPG≥126 mg/dL or were under treatment with
oral antidiabetic drugs. The subjects had no electrocar-
diogram (ECG) and/or angiography abnormalities, no
documented history of CAD and no sign of myocardial
ischemia during exercise. Other exclusion criteria for
this group were possession of type 1 diabetes mellitus,
renal disease, hepatic disease, endocrine disease, and
other metabolic diseases. also considered to be genetic determinants of plasma
lipoprotein levels and play a central role in the develop-
ment of CAD. However, it is unclear whether apoE gene
is associated with T2DM. The primary aim of this study
was to demonstrate the influence of the apoE gene poly-
morphism on plasma lipids is notable and is an impor-
tant determinant of T2DM and CAD. Secondarily, this
genetic study might also add more information about a
Thai population beyond traditional risk factors. Lipid analysis and biochemical determination The normal healthy control group consisted of 149
subjects with fasting plasma glucose (FPG) < 100 mg/dL
from among those who were randomly selected after rou-
tine health check-up to screen out those having hyperli-
pidemia, hypertension, history of chest pain, family
history of CAD or any forms of cardiovascular disease,
diabetes mellitus, hepatic and renal diseases, inflamma-
tion, general illness, traumatic injury, endocrine disease,
and other metabolic disorders. Subjects under medication
or drug abusers were also excluded. p
y
Venous blood samples were collected from the patients
after 12 hours of overnight fast. Plasma (TC), TG, HDL-
C, glucose and glycosylated hemoglobin (HbA1c) were
quantified using an automated clinical chemistry analy-
zer and enzyme-based colorimetric lists supplied by
Roche Diagnostics, Germany. LDL-C levels were calcu-
lated using Friedewald formula [24]. VLDL-C level was
obtained by using the following equation, VLDL-C =
(TC - LDL-C - HDL-C) and non-HDL-C level was Page 4 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 levels of physical activity (p = 0.030) as compared to
controls and T2DM patients. Lipid profile data demon-
strated significantly higher levels of TC, LDL-C, VLDL-
C, TG, non-HDL-C and lower level of HDL-C in T2DM
and CAD patients when compared to controls. Patients
with CAD had diabetes for a longer period (p < 0.0001)
and showed no significant difference in HbA1c (p =
0.151) as compared to the patients without CAD. Blood
glucose levels were also significantly higher in both
groups as compared to the controls (p < 0.0001). calculated by subtracting HDL-C value from total cho-
lesterol value as a candidate biometrical equivalent to
apoB 100 in diabetes [25]. levels of physical activity (p = 0.030) as compared to
controls and T2DM patients. Lipid profile data demon-
strated significantly higher levels of TC, LDL-C, VLDL-
C, TG, non-HDL-C and lower level of HDL-C in T2DM
and CAD patients when compared to controls. Patients
with CAD had diabetes for a longer period (p < 0.0001)
and showed no significant difference in HbA1c (p =
0.151) as compared to the patients without CAD. Blood
glucose levels were also significantly higher in both
groups as compared to the controls (p < 0.0001). Association of apoE gene polymorphism and diseases p
g
p
y
p
Low lipid clearance property of ε4 allele may possibly
make it an independent risk factor for the development
of CAD and T2DM. Thus, E3/E4 genotype may be the
risk factor for development of CAD and/or diabetes. The
univariate analysis was used to determine these associa-
tions according to the genotype and allele frequencies of
apoE gene polymorphism. Statistical analysis All statistical analyses were performed with IBM SPSS v1
(IBM corporation, Armonk, New York, US) and Micro-
soft 2010 based Excel (Microsoft, US). All data were
expressed as mean (SEM). One-way analysis of variance
(ANOVA) followed by posthoc Bonferroni multiple com-
parison test was applied to evaluate the mean difference
of the data between three groups (control, T2DM with-
out CAD, and T2DM with CAD). Independent samples
t-tests were applied to evaluate the mean difference in
HbA1c, diabetes duration between T2DM and T2DM
with CAD and lipid profiles between groups. Categorical
data such as sex, hypertension, history of smoking, his-
tory of alcohol drinking, physical activity and dyslipide-
mia were evaluated by Chi-square tests or Fisher’s exact
tests. Allele and genotype difference between groups and
deviations from Hardy-Weinberg equilibrium were tested
by Chi-square tests. The association between diseases
and polymorphism was provided by crude or univariable
logistic regression analysis with unadjusted odds ratio
(OR) and 95% confidence interval (95% CI). The adjusted
OR with 95% CI was used to determine the independent
risk factor for development of diabetes and CAD by mul-
tivariable logistic regression analysis after adjusting for
age, sex, BMI, smoking habits, and physical activity. The
multivariable logistic regression analysis was also
repeated for adjusted OR with 95% CI to determine the
risk of apoE polymorphism combined with obesity and
smoking (joint association) after adjusting for age, sex,
and physical activity. Statistical significance was consid-
ered as p < 0.05. ApoE genotype and allele frequencies The genotype distribution of both controls and CAD
patients were in Hardy-Weinberg equilibrium except for
T2DM that deviated from the basic norm (p = 0.0001). This was due to the lower presence of E2/E3 genotype in
diabetic group (Table 2). However, since it was unlikely
that any of the assumptions for Hardy-Weinberg equili-
brium were violated, such a departure was attributed to
chance. E3/E3 is also the most common genotype in Thai
general population. E3/E3 genotype and ε3 allele were
significantly lower (p = 0.003 and 0.010 respectively)
while E3/E4 genotype was significantly higher only in
T2DM with CAD subjects (p = 0.0004). In T2DM sub-
jects, E3/E4 genotype had a tendency to be higher
(19.35% vs. 14.09%) but showed no significant difference
(p = 0.219). In addition, E2/E3 genotype and ε2 allele
were also significantly lower in T2DM patients (p = 0.005
and 0.010, respectively). However, ε4 allele frequency
manifested itself as significantly higher in both T2DM
and CAD patients as compared to the controls (p = 0.047
and 0.009, respectively) (Table 3). The baseline characteristics of the study population The baseline characteristics of the study population
All enrolled subjects were Thais recruited from Bangkok
area. The anthropometric and demographic data were
summarized along with the clinical and biochemical
data as shown in Table 1. The data from normal con-
trols were used to compare with the data from T2DM
with and without CAD. A significant age difference was
found between control and T2DM with CAD groups
(p < 0.0001) which particularly signifies that increase in
age could possibly lead to higher chances of developing
CAD. There was also significant sex difference between
controls and CAD (p < 0.0001) due to the high preva-
lence of males among CAD patients. Other clinical data
such as BMI, systolic blood pressure (SBP) and diastolic
blood pressure (DBP) were significantly higher in both
groups when compared to controls. CAD patients had
higher frequencies of smoking (p = 0.033) and lower p
g
p y
p
Interestingly, E3/E4 genotype increased the risk of
CAD with unadjusted OR 2.78 (95%CI 1.50-5.16, p =
0.0004) and showed no association with T2DM with
unadjusted OR 1.46 (95%CI 0.76-2.81, p = 0.219). The ε4
allele appeared to increase risk of both T2DM and CAD
with unadjusted OR 1.72 (95%CI 0.97-3.06, p = 0.047)
and 2.37 (95%CI 1.36-4.15, p = 0.0009, respectively)
(Table 4). After being adjusted for age, sex, BMI, smoking
habits, and physical activity using multivariable binary
logistic regression analysis as shown in Table 5 the E3/E4
genotype appeared to be the independent risk factor for
development of CAD with adjusted OR 2.52 (95%CI
1.28-4.97, p = 0.008). However, the E3/E4 genotype was Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Chaudhary et al. The baseline characteristics of the study population not found to be an independent risk factor for diabetes,
but the ε4 allele was the independent risk factor of both
T2DM and CAD with adjusted OR 2.04 (95%CI 1.07-
3.86, p = 0.029) and OR 2.32 (95%CI 1.17-4.61, p =
0.016), respectively. The multivariable analysis of apoE4
polymorphism and the risk of T2DM and CAD were also
determined according to anthropometric and demo-
graphic characteristics. Sex (particularly male gender),
age and BMI were also independent risks for CAD but
only BMI was the independent risk for T2DM (Table 5). not found to be an independent risk factor for diabetes,
but the ε4 allele was the independent risk factor of both
T2DM and CAD with adjusted OR 2.04 (95%CI 1.07-
3.86, p = 0.029) and OR 2.32 (95%CI 1.17-4.61, p =
0.016), respectively. The multivariable analysis of apoE4
polymorphism and the risk of T2DM and CAD were also
determined according to anthropometric and demo-
graphic characteristics. Sex (particularly male gender),
age and BMI were also independent risks for CAD but
only BMI was the independent risk for T2DM (Table 5). From joint association analysis, E3/E4 genotype was found
to further increase the risk for development of diabetes
and CAD when combined with either obesity or smoking
after being adjusted for age, sex, and physical activity. The
risk factor for T2DM increased from adjusted OR 1.42
(95%CI 0.72-2.78, p = 0.310) (Table 5) to adjusted OR 4.93
(95%CI 1.74-13.98, p = 0.003) (Table 6), whereas CAD risk
increased from adjusted OR 2.52 (95%CI 1.28-4.97, p =
0.008) (Table 5) to adjusted OR 10.48 (95%CI 3.56-30.79,
P < 0.0001) (Table 6). However, the number of E3/E4 From joint association analysis, E3/E4 genotype was found
to further increase the risk for development of diabetes
and CAD when combined with either obesity or smoking
after being adjusted for age, sex, and physical activity. The
risk factor for T2DM increased from adjusted OR 1.42
(95%CI 0.72-2.78, p = 0.310) (Table 5) to adjusted OR 4.93
(95%CI 1.74-13.98, p = 0.003) (Table 6), whereas CAD risk
increased from adjusted OR 2.52 (95%CI 1.28-4.97, p =
0.008) (Table 5) to adjusted OR 10.48 (95%CI 3.56-30.79,
P < 0.0001) (Table 6). The baseline characteristics of the study population Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Page 5 of 11 Table 1 Anthropometric, demographic and clinical data of T2DM with and without CAD compared to normal healthy
controls
Variables
Controls (n = 149)
T2DM (n = 155)
T2DM + CAD (n = 147)
Age (Years)
52.01 (0.62)
51.95 (0.53)
57.56 (0.47)**
Sex (male/female)
49/100
57/98
95/52**
BMI (kg/m2)
23.84 (0.27)
26.89 (0.31)*
27.49 (0.36)**
SBP (mmHg)
111.54 (1.12)
147.31 (1.76)*
155.46 (1.96)**
DBP (mmHg)
73.11 (0.86)
84.50 (1.01)*
92.26 (1.20)**
Hypertension (%)
-
73.54
93.19
Smokers (%)
3.35
4.51
9.52†
Alcohol consumer (%)
23.80
25.50
32.70
Physical activity (%)
68.70
67.09
56.46†
Diabetes duration (Years)
-
6.09 (0.37)
9.07 (0.62)
Glucose (mg/dL)
89.7 (0.64)
225.9 (6.41)*
192.9 (6.55)**
HbA1c (%)
-
8.94 (0.16)
8.60 (0.17)
Triglyceride (mg/dL)
98.23 (3.68)
221.1 (10.35)*
204.3 (8.70)**
TC (mg/dL)
191.52 (1.80)
237.76 (5.47)*
202.48(4.19)†
LDL-C (mg/dL)
111.31(1.76)
146.65(4.84)*
122.36(3.93)†
HDL-C (mg/dL)
60.17 (1.23)
50.42 (1.27)*
44.59 (0.95)**
VLDL-C (mg/dL)
19.65 (0.73)
45.86 (2.67)*
40.86 (1.74)**
Non-HDL-C (mg/dL)
131.35 (1.95)
182.26 (4.46)*
157.88 (4.09)**
TC/HDL-C
3.4 (0.1)
4.9 (0.2)*
4.8 (0.1)**
Dyslipidemia (%)
-
85.2
100
Data are presented as mean values (SEM), or numbers (n) and percentage of subjects. *p-value < 0.0001 in comparison between controls and T2DM, **p-value <
0.0001 in comparison between controls and T2DM + CAD, †p-value < 0.05 in comparison between controls and T2DM + CAD. BMI: body mass index; SBP: systolic
blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1c; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; TG: triglyceride; HDL-C:
high density lipoprotein cholesterol; VLDL-C: very low density lipoprotein cholesterol. Table 1 Anthropometric, demographic and clinical data of T2DM with and without CAD comp
controls Table 1 Anthropometric, demographic and clinical data of T2DM with and without CAD compared to normal healthy
l raphic and clinical data of T2DM with and without CAD compared to normal healthy Data are presented as mean values (SEM), or numbers (n) and percentage of subjects. *p-value < 0.0001 in comparison between controls and T2DM, **p-value <
0.0001 in comparison between controls and T2DM + CAD, †p-value < 0.05 in comparison between controls and T2DM + CAD. BMI: body mass index; SBP: systolic
blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1c; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; TG: triglyceride; HDL-C:
high density lipoprotein cholesterol; VLDL-C: very low density lipoprotein cholesterol. Relationship between Lipid profiles and apoE4-bearing
genotypes It has been reported that dyslipidemia or dyslipoproteine-
mia might strongly contribute towards aggravating the
problems of micro- and macroangiopathic complications
in diabetic patients [26,27]. This implication was mainly
characterized by higher prevalence of male gender, older
age, smoking habits, and less physical activity. Other dif-
ferences were duration of diabetes, lower levels of HDL-C
and hypertension when compared to those without CAD
which were similar to this study. However, some studies
have also shown that elevation of TG and non-esterified
fatty acid (NEFA) levels accelerated the pathogenesis of
T2DM [28-30]. This suggests that dyslipidemia is asso-
ciated with both T2DM and CAD. Genetic factors which
have strong impact on the metabolism of plasma lipid
have been studied regarding the potential effect of T2DM
and cardiovascular outcomes in various diabetic and non-
diabetic subjects. These studies have underscored that The significant differences in apo E distribution among
these three groups are mainly due to the differences in
frequencies of the ε4 allele. These distributions in apo
ε4 allele frequencies may lead to the differences in
plasma lipid levels. To test this hypothesis, we analyzed
the correlation between apoE4 and plasma lipid levels
(Table 7). There were no significant differences between
E4 carriers (E2/E4, E3/E4, E4/E4) and E3/E3 genotype
as a reference for all values of plasma lipid levels in
both control and T2DM with CAD. In T2DM group,
there were significant elevation in the values of VLDL-C
and TG but there were no significant differences in TC,
LDL-C, HDL-C and non-HDL-C levels in E4 carriers. The baseline characteristics of the study population However, the number of E3/E4 Table 2 Frequency distribution of apoE genotypes and alleles in Hardy-Weinberg Equilibrium
Genotype
Controls (n = 149)
T2DM (n = 155)
T2DM + CAD (n = 147)
E2/E2
2 (1.34%)
1 (0.64%)
1 (0.68%)
E3/E3
113 (75.83%)
117 (75.48%)
88 (59.86%)
E4/E4
1 (0.67%)
4 (2.58%)
1 (0.68%)
E2/E3
12 (8.05%)
2 (1.29%)
11 (7.48%)
E2/E4
0 (0%)
1 (0.64%)
0 (0%)
E3/E4
21 (14.09%)
30 (19.35%)
46 (31.29%)
p-value
0.198
0.0001
0.157
Allele ε2(95% CI)
0.05(0.03 - 0.08)
0.02(0.01 - 0.04)
0.04(0.02 - 0.07)
Allele ε3(95% CI)
0.87(0.82 - 0.90)
0.86(0.81 - 0.89)
0.79 (0.74 - 0.83)
Allele ε4(95% CI)
0.07(0.05 - 0.11)
0.13 (0.09 - 0.16)
0.16 (0.12 - 0.21) Table 2 Frequency distribution of apoE genotypes and alleles in Hardy-Weinberg Equilibrium tribution of apoE genotypes and alleles in Hardy-Weinberg Equilibrium Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Page 6 of 11 Table 3 Genotype and Allele frequencies distribution of apoE gene polymorphism in controls, T2DM with and without
CAD compared to healthy controls
Genotype
Groups
Controls (n = 149)
T2DM (n = 155)
T2DM + CAD (n = 147)
E2/E2
0.013
0.006
0.006
E3/E3
0.758
0.754
0.598**
E4/E4
0.007
0.025
0.007
E2/E3
0.080
0.012*
0.074
E2/E4
0
0.006
0
E3/E4
0.140
0.193
0.321***
Allele ε2
0.05
0.02*
0.04
Allele ε3
0.87
0.86
0.79**
Allele ε4
0.07
0.13*
0.16††
*p-value < 0.01 compared between controls and T2DM, **p-value < 0.01, ***p-value < 0.001 compared between controls and T2DM + CAD. Table 3 Genotype and Allele frequencies distribution of apoE gene polymorphism in controls, T2
CAD compared to healthy controls Table 3 Genotype and Allele frequencies distribution of apoE gene polymorphism in controls, T2DM with and without
CAD compared to healthy controls
Genotype
Groups Allele frequencies distribution of apoE gene polymorphism in controls, T2DM with and without
lthy controls TG levels while HDL-C concentration was significantly
decreased as compared to E3/E3. genotypes combined with both obesity and smoking in
T2DM with and without CAD was small; thus the statisti-
cal power of this joint association comparison is limited. Relationship between Lipid profiles and apoE4-bearing
genotypes ApoE
gene is one of the most widely studied candidate genes for
CAD or any other form of cardiovascular disease and/or
diabetes. A significant relationship of apo E polymorphism
and CAD has been observed in several ethnic groups,
including Caucasian in the USA [32], Austrian [33], Fin-
nish [34], Italian [35], Turkish [36], Indian [37] and Chi-
nese [38] populations. Some studies have shown apoE ε4
allele as an independent risk factor after further adjust-
ment of other established risk factors for development of
CAD in T2DM [16] and myocardial infraction [37]
patients. However, no independent association was
observed after adjusting for age, sex, smoking, BMI, HDL-
C and TG in African- Americans and Caucasians [39]. Therefore, it is of great interest to study the independent
risk factors of this gene polymorphism in a Thai popula-
tion. The present study also coincides with a previous
study regarding the development of CAD in T2DM sub-
jects indicating that the frequency of ε4 allele is signifi-
cantly higher in CAD compared to controls and can be
one of the factors for the progression of CAD disease [40]. The frequencies of apoE allele and genotype vary between
different populations [13]. In this study, ε3 allele was the
most frequent allele in control, diabetes, and CAD sub-
jects, while ε2 was less frequent. E3/E3 genotype was the
most frequent isoform found in all groups compared to
the other genotypes which corresponds to previous reports [13,41]. Additionally, the higher frequency of E3/E4 geno-
type was observed only in CAD (p = 0.0004), whereas the
higher frequency of ε4 allele was observed in both T2DM
and CAD (p = 0.047, p = 0.0009, respectively). From this
study we can conclude that among these selected apoE
alleles (ε2, ε3, and ε4), ε4 allele is one of the predictors of
both diseases in Thai subjects. In addition, the PDAY
(Pathobiological Determinants of Atherosclerosis in
Youth) study reported that individuals with E2/E3 geno-
type had fewer atherosclerotic lesions, whereas those with
the E3/E4 had more lesions in the abdominal aorta [42]. These observations strongly suggest that the ε2 allele has a
protective role against atherosclerosis. However, in this
study, we found that E2/E3 genotype and ε2 allele were
also significantly lower in T2DM group indicating that the
protective effect of ε2 allele on the development of hyperli-
pidemia might be less in T2DM patients. Relationship between Lipid profiles and apoE4-bearing
genotypes It is probable
that other environmental and genetic factors are involved
in the pathogenesis of CAD. apoE gene encoding apolipoprotein E is associated with
significant variation in lipid profile in our body [31]. ApoE
gene is one of the most widely studied candidate genes for
CAD or any other form of cardiovascular disease and/or
diabetes. A significant relationship of apo E polymorphism
and CAD has been observed in several ethnic groups,
including Caucasian in the USA [32], Austrian [33], Fin-
nish [34], Italian [35], Turkish [36], Indian [37] and Chi-
nese [38] populations. Some studies have shown apoE ε4
allele as an independent risk factor after further adjust-
ment of other established risk factors for development of
CAD in T2DM [16] and myocardial infraction [37]
patients. However, no independent association was
observed after adjusting for age, sex, smoking, BMI, HDL-
C and TG in African- Americans and Caucasians [39]. Therefore, it is of great interest to study the independent
risk factors of this gene polymorphism in a Thai popula-
tion. The present study also coincides with a previous
study regarding the development of CAD in T2DM sub-
jects indicating that the frequency of ε4 allele is signifi-
cantly higher in CAD compared to controls and can be
one of the factors for the progression of CAD disease [40]. The frequencies of apoE allele and genotype vary between
different populations [13]. In this study, ε3 allele was the
most frequent allele in control, diabetes, and CAD sub-
jects, while ε2 was less frequent. E3/E3 genotype was the
most frequent isoform found in all groups compared to
the other genotypes which corresponds to previous reports This present study is the first report to demonstrate
that the ε4 allele containing genotypes is the major pre-
dictor of development of both T2DM and CAD. Our
study also shows strong association of E3/E4 genotype
with development of CAD in T2DM patients (Table 4) as
reported in previous studies [16,42]. †† After adjusting for age, sex and physical activity. Normal reference ranges of clinical and biochemical data are described in materials and methods. Relationship between Lipid profiles and apoE4-bearing
genotypes Table 5 Association of apoE gene polymorphism with the risk of T2DM and CAD compared to healthy controls
represented as adjusted OR Table 5 Association of apoE gene polymorphism with the risk of T2DM and CAD compared
represented as adjusted OR tion of apoE gene polymorphism with the risk of T2DM and CAD compared to healthy controls
adj sted OR apoE gene encoding apolipoprotein E is associated wi
significant variation in lipid profile in our body [31]. Apo
gene is one of the most widely studied candidate genes f
CAD or any other form of cardiovascular disease and/
diabetes. A significant relationship of apo E polymorphis
and CAD has been observed in several ethnic group
including Caucasian in the USA [32], Austrian [33], Fi
nish [34], Italian [35], Turkish [36], Indian [37] and Ch
nese [38] populations. Some studies have shown apoE
allele as an independent risk factor after further adjus
ment of other established risk factors for development
CAD in T2DM [16] and myocardial infraction [3
patients. However, no independent association w
observed after adjusting for age, sex, smoking, BMI, HD
C and TG in African- Americans and Caucasians [39
Therefore, it is of great interest to study the independe
risk factors of this gene polymorphism in a Thai popul
tion. The present study also coincides with a previo
study regarding the development of CAD in T2DM su
jects indicating that the frequency of ε4 allele is signi
cantly higher in CAD compared to controls and can b
one of the factors for the progression of CAD disease [40
The frequencies of apoE allele and genotype vary betwe
different populations [13]. In this study, ε3 allele was th
most frequent allele in control, diabetes, and CAD su
jects, while ε2 was less frequent. E3/E3 genotype was th
most frequent isoform found in all groups compared
the other genotypes which corresponds to previous repor
Table 6 Joint association study to evaluate the risk of T
Genotype
Obesity/Smoking
Controls
T2DM
Adjusted OR
E3/E3
Neither
70
34
1
E3/E3
Either
40
73
3.65 (2.07
E3/E3
Both
2
10
10.05 (2.06
E3/E4
Neither
14
14
1.97 (0.84
E3/E4
Either
6
15
4.93 (1.74
E3/E4
Both
6
1
-
†† After adjusting for age, sex and physical activity. Normal reference range apoE gene encoding apolipoprotein E is associated with
significant variation in lipid profile in our body [31]. Relationship between Lipid profiles and apoE4-bearing
genotypes After pooling the overall subjects, the results showed
that E4 carrier has significantly elevated VLDL-C and Table 4 Associations of apoE gene polymorphisms with the risk of T2DM and CAD compared to healthy controls
represented as unadjusted OR
Genotype
Controls
(n = 149)
T2DM
(n = 155)
Unadjusted OR
(95% CI)
p-value
T2DM + CAD
(n = 147)
Unadjusted OR
(95% CI)
p-value
E2/E2
2
1
0.48 (0.02 - 6.78)
0.616
1
0.50 (0.02 - 7.16)
1.000
E3/E3
113
117
0.98 (0.56 - 1.71)
0.942
88
0.48 (0.28 - 0.81)
0.003
E4/E4
1
4
3.92 (0.41 - 93.19)
0.371
1
1.01(0.06 - 16.36)
1.000
E2/E3
12
2
0.15 (0.02 - 0.72)
0.005
11
0.92(0.36 - 2.33)
0.854
E2/E4
0
1
-
1.000
0
-
-
E3/E4
21
30
1.46 (0.76 - 2.81)
0.219
46
2.78(1.50 - 5.16)
0.0004
Allele ε2
16
5
0.29 (0.09 - 0.85)
0.011
13
0.81(0.36 - 1.81)
0.580
Allele ε3
259
266
0.91 (0.56 - 1.48)
0.691
234
0.57(0.36 - 0.90)
0.107
Allele ε4
23
39
1.72 (0.97 - 3.06)
0.047
49
2.37(1.36 - 4.15)
0.0009 Table 4 Associations of apoE gene polymorphisms with the risk of T2DM and CAD compared to healthy controls
represented as unadjusted OR ns of apoE gene polymorphisms with the risk of T2DM and CAD compared to healthy controls
adjusted OR ble 4 Associations of apoE gene polymorphisms with the risk of T2DM and CAD compared to hea
presented as unadjusted OR Page 7 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Table 5 Association of apoE gene polymorphism with the risk of T2DM and CAD compared to healthy controls
represented as adjusted OR
T2DM
T2DM + CAD
Adjusted OR (95% CI)††
p-value
Adjusted OR (95% CI)††
p-value
E3/E4 genotype
1.42 (0.72 - 2.78)
0.310
2.52 (1.28 - 4.97)
0.008††
ε4 allele
2.04 (1.07 - 3.86)
0.029††
2.32 (1.17 - 4.61)
0.016††
Age
1.00 (0.97 - 1.04)
0.887
1.13 (1.08 - 1.18)
<0.0001
Sex
1.29 (0.77 - 2.14)
0.326
3.10 (1.75 - 5.49)
<0.0001
BMI
3.68 (2.25 - 6.00)
<0.0001
3.66 (2.09 - 6.39)
<0.0001
Smoking
2.58 (0.87 - 7.66)
0.086
0.59 (0.13 - 2.50)
0.473
Physical activity
0.68 (0.40 - 1.19)
0.148
0.67 (0.36 - 1.24)
0.206
††After adjusting for age, sex, BMI, smoking habit and physical activity. Normal reference ranges of clinical and biochemical data are described in materials and
methods. E3
(n
206.5
125.3
45.49
41.25
206.26
161.0
E4-bea s
and s
and 45.49
41.25
6.26
61.0
-bea E3
(n =
206.5
125.3
45.49
41.25
206.26
161.0
E4-bea Relationship between Lipid profiles and apoE4-bearing
genotypes After adjusting for
age, sex, smoking, BMI, and physical activity, E3/E4 con-
taining subjects showed 2.52-fold higher risk (p = 0.008)
while ε4 allele containing genotypes led to a 2.32-fold Table 6 Joint association study to evaluate the risk of T2DM and CAD compared to controls
Genotype
Obesity/Smoking
Controls
T2DM
Adjusted OR (95% CI)††
p-value
T2DM + CAD
Adjusted OR (95% CI)††
p-value
E3/E3
Neither
70
34
1
-
30
1
-
E3/E3
Either
40
73
3.65 (2.07 - 6.43)
<0.0001
56
2.24 (1.15 - 4.35)
0.018
E3/E3
Both
2
10
10.05 (2.06 - 48.96)
0.004
1
2.02 (0.16 - 25.01)
0.585
E3/E4
Neither
14
14
1.97 (0.84 - 4.64)
0.118
7
1.02 (0.34 - 3.06)
0.970
E3/E4
Either
6
15
4.93 (1.74 - 13.98)
0.003
36
10.48 (3.56 - 30.79)
<0.0001
E3/E4
Both
6
1
-
-
3
-
-
†† Table 6 Joint association study to evaluate the risk of T2DM and CAD compared to controls T2DM + CAD
3/E3
E4-bearing genotypes
E
= 88)
(n = 47)
(n
52 (5.26)
192.04 (7.96)
210.8
33 (4.84)
113.80 (7.33)
127.4
49 (1.27)
42.70 (1.37)
52.6
25 (2.37)
38.91 (3.04)
33.7
26 (11.78)
194.53 (15.21)
168.8
03(5.24)
149.34(7.44)
158.2
aring genotypes. Data are presented as mean va (n
210
12
52
33
168
158
mean Page 9 of 11 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 allele and lipid profile is still controversial [51]. The ε4
allele has been shown to be associated with high concen-
tration of serum TC and LDL-C in Chinese population
[38]. Some studies also showed a significant relationship
between E3/E4 genotype with lower HDL-C and higher
LDL-C concentrations in CAD patients [52] and with
higher TG levels in T2DM patients [48] as compared to
the healthy controls. However, a study in the Tunisian
population has suggested that HDL-C concentration and
other lipid profiles are not associated with apoE gene poly-
morphism in the total population studied, but has proven
that ε4 allele increased LDL-C in type 2 diabetic men [53]. Association of ε4 allele with higher LDL-C and lower
HDL-C levels was only found in type 2 diabetic women in
Spanish population [54] indicating that gender affects the
effect of apoE gene polymorphism. Relationship between Lipid profiles and apoE4-bearing
genotypes In addition, incidence of T2DM is also subject to
gene-environment interactions, for example, dietary fac-
tors, intake of vegetable fat, polyunsaturated fatty acid,
dietary fiber (particularly cereal fiber), magnesium, and
caffeine were significantly inversely correlated and
intakes of trans fat, saturated fatty acid and heme-iron,
glycemic index, and glycemic load were significantly posi-
tively correlated with the incidence of T2DM [43]. Simi-
larly, regular exercise has been shown to reduce weight,
BMI, HDL-C and insulin resistance. This indicates that
exercise is also associated with decrease risk of T2DM
and CAD. Furthermore, apoE gene polymorphism has
been shown to modulate the effects of exercise on lipo-
protein concentrations in plasma [44]. In this study,
smoking and obesity are shown to be the two major con-
tributing factors that can promote the development of
T2DM and CAD or both when placed in joint association
(Table 6). The reason for this is that smoking and obesity
enhance the oxidative stress which results in decreased
insulin secretion from pancreatic b-cell and decreased
uptake of blood glucose into the muscle cells [9,10]. This
evidence was supported by the experimental study on
atherosclerosis-susceptible B6 (B6.apoE -/-) and athero-
sclerosis-resistant BALB (BALB.apoE -/-) mice that
showed defects in insulin secretion rather than defects in
insulin resistance which explains the mark difference in
susceptibility to T2DM [45]. Moreover, oxidative stress
also increases vascular inflammation leading to CAD
and/or any form of cardiovascular disease with or with-
out combination of T2DM [46]. To demonstrate that apoE gene polymorphism is
involved in the lipid clearance process and it has great
influence on the lipid level in our body [47,48]. Although
previous study has shown that non-HDL-C concentration
is similar to or better than LDL-C alone in predicting car-
diovascular disease (CVD) incidence [49], in this study, the
results showed that the decrease in HDL-C and elevation
of TG, VLDL-C and LDL-C levels (Table 1) are also the
landmarks of diabetes and CAD development as found in
an earlier study [50]. However, the association of apoE ε4 Relationship between Lipid profiles and apoE4-bearing
genotypes In this study, there is a
mean comparison between E4 carriers and E3/E3 geno-
type in the T2DM and total population studied which
showed a significantly higher VLDL-C, TG and lower
HDL-C levels in E4 carriers (Table 7) similar to the report
of Knouff et al 1999 [15]. Nevertheless, the differences of
lipid profiles were not observed in controls and T2DM
with CAD. This may be due to dietary restrictions in the
controls and lipid-lowering aggressive treatment in T2DM
with CAD. Kolovou et al. 2006 [55] reported that the ε4
allele can increase LDL-C concentrations in the presence
of an atherogenic diet, but a lower fat diet can suppress
this effect. On the other hand, aerobically trained indivi-
duals have high HDL and display enhanced glucose toler-
ance [56]. A lipid-lowering-drug which raises HDL levels
and decrease TG levels delays the onset of T2DM and
reduces the development of atherosclerosis [57]. In addi-
tion, loss of caspase-1 activity which involves the inflam-
matory process in human atherosclerotic vessel has shown
to reduce atherosclerosis lesion formation in Casp1-/- Apo
E-/- mice [58]. Another reason for these variations of lipid
profiles might be because of the differences in genetic
background and the prevalence of high oxidative stress in
the studied population. When considering the basis of
gene-gene and gene-environment interactions, as
described above, the polymorphism study at the genome
level might not provide a real picture for development of
diseases in respect to lipid profile. A more robust parallel
study at the protein level, or of other candidate genes, may
be necessary to evaluate the mechanism of T2DM and
CAD development. However, this study will provide a
good starting point for the screening of large populations
before proceeding to the protein level study of apoE and
other candidate genes in various races and, additionally,
including pharmacogenomics study in the future. higher risk (p = 0.016) for developing CAD and ε4 allele
containing genotypes led to a 2.04-fold higher risk (p =
0.029) in the development of T2DM as compared to the
controls. This signifies that other risk factors have no
influence in development or curbing the disease, suggest-
ing that ε4 allele is the independent risk factor for both
T2DM and CAD. However, E3/E4 genotype further
increases the risk for the development of diabetes and
CAD when combined with smoking and/or obesity
(Table 6). Relationship between Lipid profiles and apoE4-bearing
genotypes This confirms the findings that progression
and development of diabetes and CAD is the conse-
quence of multifactorial parameters, for example, obesity,
smoking, oxidative stress, defect in pancreatic b-cells,
alcohol consumption, exercise, hypertension and genetic
factors. In addition, incidence of T2DM is also subject to
gene-environment interactions, for example, dietary fac-
tors, intake of vegetable fat, polyunsaturated fatty acid,
dietary fiber (particularly cereal fiber), magnesium, and
caffeine were significantly inversely correlated and
intakes of trans fat, saturated fatty acid and heme-iron,
glycemic index, and glycemic load were significantly posi-
tively correlated with the incidence of T2DM [43]. Simi-
larly, regular exercise has been shown to reduce weight,
BMI, HDL-C and insulin resistance. This indicates that
exercise is also associated with decrease risk of T2DM
and CAD. Furthermore, apoE gene polymorphism has
been shown to modulate the effects of exercise on lipo-
protein concentrations in plasma [44]. In this study,
smoking and obesity are shown to be the two major con-
tributing factors that can promote the development of
T2DM and CAD or both when placed in joint association
(Table 6). The reason for this is that smoking and obesity
enhance the oxidative stress which results in decreased
insulin secretion from pancreatic b-cell and decreased
uptake of blood glucose into the muscle cells [9,10]. This
evidence was supported by the experimental study on
atherosclerosis-susceptible B6 (B6.apoE -/-) and athero-
sclerosis-resistant BALB (BALB.apoE -/-) mice that
showed defects in insulin secretion rather than defects in
insulin resistance which explains the mark difference in
susceptibility to T2DM [45]. Moreover, oxidative stress
also increases vascular inflammation leading to CAD
and/or any form of cardiovascular disease with or with-
out combination of T2DM [46]. higher risk (p = 0.016) for developing CAD and ε4 allele
containing genotypes led to a 2.04-fold higher risk (p =
0.029) in the development of T2DM as compared to the
controls. This signifies that other risk factors have no
influence in development or curbing the disease, suggest-
ing that ε4 allele is the independent risk factor for both
T2DM and CAD. However, E3/E4 genotype further
increases the risk for the development of diabetes and
CAD when combined with smoking and/or obesity
(Table 6). This confirms the findings that progression
and development of diabetes and CAD is the conse-
quence of multifactorial parameters, for example, obesity,
smoking, oxidative stress, defect in pancreatic b-cells,
alcohol consumption, exercise, hypertension and genetic
factors. Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 Chaudhary et al. Cardiovascular Diabetology 2012, 11:36
http://www.cardiab.com/content/11/1/36 without CAD complication by influencing the plasma
lipid levels that are important risk factors for both
T2DM and CAD. No single factor can give a satisfactory
explanation regarding development of diabetes and CAD
as both diseases are complex diseases. Focusing, there-
fore, on only one risk factor may be less than optimal in
enabling researchers to predict who will develop unto-
ward events in complex diseases like diabetes and CAD. Nonetheless, our study strongly supported that the apoE
ε4 allele is an independent risk factor for development
of both T2DM and CAD. Moreover, obesity and/or
smoking, conditions associated with high oxidative
stress, can aggravate the progression of both diseases. Genetic studies can thus provide information that may
help to improve the ability to identify individuals,
families and populations at increased risk, as well as to
improve the clinical management of patients with
T2DM and CAD. Such information may be useful in
developing public health programs reinforcing primary
and secondary prevention for T2DM and CAD. Further-
more, T2DM and CAD patients identified to carry high
risk genotype or allele should be treated aggressively to
prevent the progression of disease. Abbreviations
APOE A
li 8. Boden G, Shulman GI: Free fatty acids in obesity and type 2 diabetes:
defining their role in the development of insulin resistance and beta-cell
dysfunction. Eur J Clin Invest 2002, 32(Suppl 3):14-23. 8. Boden G, Shulman GI: Free fatty acids in obesity and type 2 diabetes:
defining their role in the development of insulin resistance and beta-cell
dysfunction. Eur J Clin Invest 2002, 32(Suppl 3):14-23. APOE: Apolipoprotein E; CAD: Coronary artery disease; T2DM: Type 2
diabetes mellitus; PCR-RFLP: Polymerase chain reaction-restriction fragment
length polymorphism; HDL-C: High-density lipoprotein cholesterol; VLDL-C:
Very low density lipoprotein cholesterol; LDL-C: Low-density lipoprotein
cholesterol; TC: Total cholesterol; TG: Triglyceride; non-HDL-C: Non-high-
density lipoprotein cholesterol; SNPs: Single nucleotide polymorphisms; NF-
κB: Nuclear factor kappa B; LDLR: Low density lipoprotein receptor; T:
Thymine; C: Cytosine; ECG: Electrocardiogram; WHO: World health
organization; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; BMI:
Body mass index; FPG: Fasting plasma glucose; Guanidine-HCL: Guanidine-
hydrochloride; APO B-100: Apolipoprotein B-100; HbA1c: Glycated
hemoglobin A1c; NEFA: Non-esterified fatty acid; PDAY: Pathobiological
determinants of atherosclerosis in youth. 9. Martyn JA, Kaneki M, Yasuhara S: Obesity-induced insulin resistance and
hyperglycemia: etiologic factors and molecular mechanisms. Anesthesiology 2008, 109:137-148. 10. Evans JL, Goldfine ID, Maddux BA, Grodsky GM: Oxidative stress and
stress-activated signaling pathways: a unifying hypothesis of type 2
diabetes. Endocr Rev 2002, 23:599-622. 11. Grundy SM: Drug therapy of the metabolic syndrome: minimizing the
emerging crisis in polypharmacy. Nat Rev Drug Discov 2006, 5:295-309. 12. Mahley RW, Rall SC Jr: Apolipoprotein E: far more than a lipid transport
protein. Annu Rev Genomics Hum Genet 2000, 1:507-537. 13. Singh PP, Singh M, Mastana SS: APOE distribution in world populations
with new data from India and the UK. Ann Hum Biol 2006, 33:279-308. Author details
1
f 1Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol
University, Bangkok 10700, Thailand. 2Department of Medicine, Faculty of
Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 3Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol
University, Bangkok 10700, Thailand. 4Department of Preventive and Social
Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok
10700, Thailand. 5Her Majesty’s Cardiac Center, Faculty of Medicine Siriraj
Hospital, Mahidol University, Bangkok 10700, Thailand. 16. Guang-da X, You-ying L, Zhi-song C, Yu-sheng H, Xiang-jiu Y:
Apolipoprotein e4 allele is predictor of coronary artery disease death in
elderly patients with type 2 diabetes mellitus. Atherosclerosis 2004,
175:77-81. 17. Wilson PW, Schaefer EJ, Larson MG, Ordovas JM: Apolipoprotein E alleles
and risk of coronary disease. A meta-analysis. Arterioscler Thromb Vasc Biol
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laboratory medicine. Clin Chem 1995, 41:1068-1086. This study was supported by the Graduate Thesis Scholarship grant from
Faculty of Medicine Siriraj Hospital, Mahidol University. The authors wish to
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lipid levels that are important risk factors for both
T2DM and CAD. No single factor can give a satisfactory
explanation regarding development of diabetes and CAD
as both diseases are complex diseases. Focusing, there-
fore, on only one risk factor may be less than optimal in
enabling researchers to predict who will develop unto-
ward events in complex diseases like diabetes and CAD. Nonetheless, our study strongly supported that the apoE
ε4 allele is an independent risk factor for development
of both T2DM and CAD. Moreover, obesity and/or
smoking, conditions associated with high oxidative
stress, can aggravate the progression of both diseases. Genetic studies can thus provide information that may
help to improve the ability to identify individuals,
families and populations at increased risk, as well as to
improve the clinical management of patients with
T2DM and CAD. Such information may be useful in
developing public health programs reinforcing primary
and secondary prevention for T2DM and CAD. Further-
more, T2DM and CAD patients identified to carry high
risk genotype or allele should be treated aggressively to
prevent the progression of disease. Received: 26 March 2012 Accepted: 23 April 2012
Published: 23 April 2012 Conclusions This study tentatively supports the fact that variability in
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2004, 88:897-909, x. 48. Authors’ contributions All authors fulfill the criteria for authorship. RC carried out the SNPs analysis. AL conceived all of the study and coordination. RC and AL conducted
statistical analysis and drafted manuscript. TP, DT, SR, and CS provided the
samples for SNP analysis. AL, TP, DT, SS, and CS participated in study
conception and design, interpretation of data and critical revision of
manuscript for important intellectual content. All authors read and approved
the final version of the manuscripts. All authors fulfill the criteria for authorship. RC carried out the SNPs analysis. AL conceived all of the study and coordination. RC and AL conducted
statistical analysis and drafted manuscript. TP, DT, SR, and CS provided the
samples for SNP analysis. AL, TP, DT, SS, and CS participated in study
conception and design, interpretation of data and critical revision of
manuscript for important intellectual content. All authors read and approved
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Qiwei granules alleviates podocyte lesion in kidney of diabetic KK-Ay mice
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BMC complementary and alternative medicine
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cc-by
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Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97
DOI 10.1186/s12906-015-0603-x Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97
DOI 10.1186/s12906-015-0603-x Open Access Abstract Background: Chinese medicine comprised of all natural herbs is widespread used in the treatment of diabetic
nephropathy (DN). Podocyte contributes to the integrity of glomerular filtration barrier whose injury plays an
important role in the initiation and progression of DN. Our study aimed to investigate the effect of Qiwei granules
on podocyte lesion in diabetic KK-Ay mice kidney and its underlying mechanism. Methods: Twelve-week-old male KK-Ay mice were randomly divided in vehicle group and Qiwei granules group,
while C57BL/6J mice were used as normal control. The mice were gavage with 1.37 g/kg/day Qiwei granules or
water for 10 weeks. We measured water, food intake and body weight (BW) and fasting blood glucose (FBG) every
2 weeks, and urine protein every 4 weeks. At the end of the experiment, all surviving mice were sacrificed. The
kidney weight and serum renal parameters were measured, and the renal morphology was observed. To search
the underlying mechanism, we examined the podocyte positive marker, slit diaphragm protein expression and
some involved cell signal pathway. Results: Qiwei granules treatment significantly improved the metabolic parameters, alleviated the urinary protein,
and protected renal function in KK-Ay mice. In addition, the glomerular injuries and podocyte lesions were mitigated
with Qiwei granules treatment. Furthermore, Qiwei granules increased expression of nephrin, CD2AP, and integrin
alpha3beta1 in the podocytes of KK-Ay mice. Qiwei granules improved the phosphoration of Akt and inhibited cleaved
caspase-3 protein expression. Conclusion: These finding suggest that Qiwei granules protects the podocyte from the development of
DN via improving slit diaphragm (SD) molecules expression and likely activating Akt signaling pathway
in KK-Ay mice. Keywords: Qiwei granules, Diabetic nephropathy, KK-Ay mice, Podocyte, Slit diaphragm, Akt si Keywords: Qiwei granules, Diabetic nephropathy, KK-Ay mice, Podocyte, Slit diaphragm, Akt signa the initiation and progression of DN [3,4]. Podocytes
are terminally differentiated and highly specialized
cells with foot processes attaching on glomerular base-
ment membrane (GBM) and interconnected by slit dia-
phragm (SD). Podocytes have function to establish the
filtration barrier, and remodel the GBM [5]. Nephrin,
one transmembrane protein component of SD, is es-
sential to maintain normal podocyte structure and
control the filtration barrier [6]. CD2AP, another trans-
membrane protein locates at podocyte slit membrane
interacting with nephrin to maintain the structure of
podocyte [7]. A number of studies suggest that both
the protein and mRNA expression of nephrin were sig-
nificantly decreased in diabetic nephropathy patients Qiwei granules alleviates podocyte lesion in
kidney of diabetic KK-Ay mice Jingxin Zhou1,2, Wen Sun1, Hisae Yoshitomi3, Linyi Li1, Lingling Qin1, Xiangyu Guo1, Lili Wu1, Yan Zhang1,
Xinli Wu1, Tunhai Xu1, Ming Gao3 and Tonghua Liu1* © 2015 Zhou et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. * Correspondence: thliu@vip.163.com
1Beijing University of Chinese Medicine, 11 North 3rd-ring East Road, 100029
Chaoyang District, Beijing, People’s Republic of China
Full list of author information is available at the end of the article Qiwei granules Qiwei
granules
were
prepared
by
Shanxi
JinYu
Pharmaceutical Company., Ltd. Qiwei granules con-
sists of Astragalus membranaceus, Rehmannia gluti-
nosa, Prunella vulgaris, Curcuma zedoaria, Euonymus
alatus, Panax pseudoginseng, and Rheum officinale. Reagents The antibodies against integrin beta1 (integrin β1), pAkt
(Ser473), Akt, cleaved caspase-3 and caspase-3 were
purchased from Cell Signaling Technology, Inc. (Beverly,
USA). Nephrin and integrin alpha3 (integrin α3) anti-
bodies were purchased from R&D Systems, Inc. (MN,
USA). CD2 associated protein antibody (CD2AP) was pur-
chased from Abcam, Inc. (MA, USA). In-situ cell death
detection kit POD was purchased from Roche Diagnositcs
(Mannheim, Germany). Wilms tumor −1 antibody (WT-1)
was purchased from Santa Cruz Biotechnology, Inc. (Texas,
USA). Chinese medicine comprised of all natural products is
widespread used in the treatment of diabetes and its
complications, which have been proved to alleviate the
progression of diabetic nephropathy [14]. Qiwei granules
(used name Zhi xiao tong mai ning) is composed of
Astragalus membranaceus, Rehmannia glutinosa, Prunella
vugaris, Curcuma zedoaria, Euonymus alatus, Panax pseu-
doginseng, and Rheum officinale. It has been reported that Astragalus membranaceus,
and Euonymus alatus both protects diabetic nephropa-
thy kidney in animals [15,16], Rehmannia glutinosa de-
creases the glucose in diabetic rats [17] and Prunella
vulgaris [18] improves diabetes and its complications
in vivo and vitro [19,20]. Many studies have been sug-
gested that Panax notoginseng improves glucose and
lipid metabolism, prevents oxidation, and ameliorates
urinary albumin in patients with chronic renal failure
[21,22]. Previous clinical observation has shown that
Qiwei granules decreased blood glucose and reduced
the proteinuria to prevent diabetic nephropathy [23]. It
also has been demonstrated that Qiwei granules pre-
vented the progression of tubulointerstitial fibrosis in
KK-Ay diabetes mice by TGF-β1/Smads signaling [24]. But the underlying mechanism on podocyte was not
unclear. Background Diabetic nephropathy (DN), one of most common and
serious complication of diabetes, is a major cause of
end-stage renal disease (ESRD) [1]. Diabetic nephropa-
thy is characterized with expansion of cellar and matrix
components in the mesangium, thickness of glomeru-
lar basement membrane leading to glomerulosclerosis
followed by persistent albuminuria and progressive
renal dyfunction [2]. However, much attention has
been focused on the importance of podocyte injury in * Correspondence: thliu@vip.163.com
1Beijing University of Chinese Medicine, 11 North 3rd-ring East Road, 100029
Chaoyang District, Beijing, People’s Republic of China
Full list of author information is available at the end of the article Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 2 of 11 Page 2 of 11 Page 2 of 11 Methods and animals [8,9]. However, some evidence indicates
that SD maintains podocyte functional integrity not
only because of foundation for the filter barrier but
also participation in cell signaling such as PI3K/Akt
[10]. Intergrins are transmembrane molecules assembled
of α and β subunits adhered on GBM to regulate the
shape and adhesion of the podocytes [11]. The integrin
α3β1 in the podocyte foot processes interacted podocyte-
GBM mediates cell adhesion and is associated with integ-
rin signaling in charge of glomerular filtration barrier. It
has been reported that expression of integrin α3β1 on
podocytes are reduced in diabetes patients and rats [12]. There are no podocyte foot processes in the mice born
lack of integrin α3 [13]. Animals BMC Complementary and Alternative Medicine (2015) 15:97 Blood and proteinuria analysis 10 min and supernatants were isolated. Proteins were
extracted by boiling in 0.5 mmol/ml Tris–HCl, PH 6.8,
10% SDS, 20% glycerol, 0.05% bromophenol blue, and
2-mercaptethanol. The proteins (40 μg/sample) were
electrophoresed on a 7.5%-12.5% sodium dodecyl sulfatepo-
lyacrylamide gel electrophoresis (SDS-PAGE) at 100 V for
2 h and transferred onto polyvinylidene fluoride (PVDF)
membranes (Merck Millipore, Germany) at 30 mA for 2 h. The membrane was blocked in 5% skim milk in Tris-
buffered saline containing 0.1% Tween 20 for 1 h, and incu-
bated with the primary antibody nephrin (1:1000), CD2AP
(1:2000), integrin α3 (1:1000), integrin β1 (1:500), pAkt
(1:1000), Akt (1:1000), cleaved caspase-3 (1:1000) and
caspase-3 (1:1000) overnight. After washing with TBST
and incubated for 1 h with horseradish peroxidase-linked
anti-rabbit and anti-mouse secondary antibodies (ZSGB-Bio,
Inc., Beijing, China). Detection was achieved using ECL kit
(Santa Cruz, USA). GAPDH was used as an internal con-
trol. The density of the bands was measured using IPP. 10 min and supernatants were isolated. Proteins were
extracted by boiling in 0.5 mmol/ml Tris–HCl, PH 6.8,
10% SDS, 20% glycerol, 0.05% bromophenol blue, and
2-mercaptethanol. The proteins (40 μg/sample) were
electrophoresed on a 7.5%-12.5% sodium dodecyl sulfatepo-
lyacrylamide gel electrophoresis (SDS-PAGE) at 100 V for
2 h and transferred onto polyvinylidene fluoride (PVDF)
membranes (Merck Millipore, Germany) at 30 mA for 2 h. The membrane was blocked in 5% skim milk in Tris-
buffered saline containing 0.1% Tween 20 for 1 h, and incu-
bated with the primary antibody nephrin (1:1000), CD2AP
(1:2000), integrin α3 (1:1000), integrin β1 (1:500), pAkt
(1:1000), Akt (1:1000), cleaved caspase-3 (1:1000) and
caspase-3 (1:1000) overnight. After washing with TBST
and incubated for 1 h with horseradish peroxidase-linked
anti-rabbit and anti-mouse secondary antibodies (ZSGB-Bio,
Inc., Beijing, China). Detection was achieved using ECL kit
(Santa Cruz, USA). GAPDH was used as an internal con-
trol. The density of the bands was measured using IPP. The blood was collected and sera were prepared by cen-
trifugation, and measured by Beckman coulter. Serum
urine nitrogen (BUN) and serum creatinine (Scr) were
measured
using
the
corresponding
commercial
kit
(Leadman, Beijing). 24 h urinary samples were collected
from mice in metabolic cages without restriction of
water intake at the 0, 4, 8 weeks of treatment. After col-
lection, urine volume was recorded and determined of
24 h urinary protein by Bradford method. Immunohistochemical staining for TUNEL and WT-1 TUNEL was performed with the In-situ cell death detec-
tion kit POD to detect apoptotic positive cells. Staining
for podocytes in renal tissues was performed using the
protocol of polyclonal rabbit anti-mouse WT-1 antibody
(1:200). Negative control for immunohistochemistry was
run incubating with nonimmune serum instead of the
primary antibody. For quantitative determination of podo-
cyte numbers, the WT-1 positive cells were calculated in a
total of 40 glomeruli area by Image J software. The aver-
ages of podocyte numbers per glomerulus were assessed
from individual five mice each group. Histological analysis Parts of kidney tissue samples were fixed in 4% parafor-
maldehyde and embedded in paraffin. Under light mi-
croscopy, 2-3 μm sections were prepared and stained
with hematoxylin and eosin (HE), Masson’s Trichrome,
and periodic acid-Schiff (PAS). For PAS stain, the
mesangial matrix expansion was evaluated in 10 ran-
domly selected glomeruli in the cortex per animal under
high magnification (×400) with Image-Pro Plus 6.0
(Media Cyemetics, Siliver Spring, MD). The area of
fibrosis was investigated by measuring the collagen de-
position staining by Masson’s Trichrome under × 200
magnification using Image-Pro Plus 6.0 software. 1 mm3
samples of renal cortex were also fixed in 2.5% glutaral-
dehyde solution, rinsed in phosphate buffer, fixed in
osmic acid and embedded in epon. Semithin 1 μm sec-
tions were cut and stained with uranyl acetate and then
lead in nitrate solution. Representative glomeruli from 5
mice, each from untreated KK-Ay mice, KK-Ay mice treated
with Qiwei granules, and normal control mice were exam-
ined. Images were acquired on a JEOL JEM-2100 transmis-
sion electron microscope (JEOL, Tokyo, Japan). Western blotting analysis The kidney tissue was homogenized with ice-cold ho-
mogenized buffer containing 50 mM Tris–HCl (8.0),
150 mM NaCl, 0.025% NaN3, 0.1% SDS, 0.1 mg/ml
PMSF, 0.01 mg/ml Aprotintin, 0.5% Na deoxycholate,
and 0.1%Nonidet-P40. After incubation on ice for
20 min, lysates were centrifuged at 10000 rpm for Statistical analysis Data are expressed as mean ± SEM and statistically ana-
lyzed by Dunnett test. Differences were considered sig-
nificant for P < 0.05. Animals Eight-week-old male KK-Ay mice and C57BL/6J mice
were purchased from institute of Laboratory Animal
Science of Chinese Academy of Medical Sciences (Beijing,
China). All mice were housed in same controlled condi-
tions under temperature of 25 ± 1°C and humidity of
55% ± 5% on a regular 12 h light/dark cycle with free
access to food and water. The KK-Ay mice were fed
with high-fat diets (458 kcal/100 g, containing 10% fat),
and C57BL/6J mice were fed with normal diets. After
acclimating to new surroundings for 4 weeks, KK-Ay
mice were randomly divided into two groups of 6 mice
each, Qiwei granules group and vehicle group, while
C57BL/6J mice (n = 6) were used as normal control
group. The mice were gavage with 1.37 g/kg/day Qiwei
granules (Qiwei granules group) or same volume water
(vehicle group and normal control group) as administra-
tion. Water, food intake and body weight (BW) of mice
and fasting blood glucose (FBG) levels were measured
every 2 weeks. All mice were placed into metabolic
cages for urine collection at 0, 4, 8 weeks of treatment. After 10 weeks of treatment, all surviving mice were
fasted for 12 h and sacrificed. The kidney were removed,
cleaned for measurement of right kidney weight, and
promptly frozen in liquid nitrogen. And portions were
appropriated in 4% paraformaldehyde for histological
analysis and in 2.5% glutaraldehyde solution for elec-
tron microscopic examination. KK-Ay mouse was chosen as a diabetic nephropathy
model in our study. KK-Ay mouse is established by
transfection of the yellow obese gene (Ay) into moderate
KK mouse [25]. KK-Ay mouse is spontaneously devel-
oped and characterized of T2 Diabetes such as obesity,
hyperglycemia, insulin resistance which is widely used as
a T2D model [26]. Furthermore, KK-Ay mouse over
12 weeks age appears albuminuria, and diffuse mesangial
matrix expansion, segmental glomerulosclerosis [27]. Therefore, KK-Ay mice are considered as good diabetic
nephropathy models renal lesions of which are similar
with those of human. The study was approved by the Ethical Committee of
Beijing University of Chinese Medicine, which was car-
ried out in accordance with the Principles of Laboratory
Animal Care published by National Institutes of Health. In the present study, we investigate whether Qiwei gran-
ules protect the podocyte and the underlying mechanism
in the KK-Ay diabetic mice kidneys. Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 3 of 11 Zhou et al. Quantitative real time RT-PCR Total RNA was extracted from the kidney using Trizol
kit (Invitrogen, USA). Then RNA (1 μg) from each sam-
ple was reverse-transcribed to cDNA using the M-MLV
RT Kit, according to the manufacturer’s instructions
(Takara). THUNDERBIRD SYBR qPCR Mix was used
for quantitative real-time RT-PCR analysis of each gene’s
expression. The primers are listed as follow: nephrin
forward, 5′- TCTGGCGGAGAAGACTGAG -3′; nephrin
reverse, 5′- GTGCTAACCGTGGAGCTTCT -3′; integrin
α3 forward, 5′- CTGGAGTGGCCCTATGAAGT -3′; in-
tegrin α3 reverse, 5′- TGACTCCAGGGTCAGAGAGA -3′;
integrin β1 forward, 5′- CTTGCTGCTGATTTGGAA
AC -3′; integrin β1 reverse, 5′- CTTCGGATTGACCAC
AGTTG -3′; GAPDH forward, 5′- GCAAGTTCAACG
GCACAG
-3′;
GAPDH
reverse,
5′-
CGCCAGTAG
ACTCCACGAC -3′. Amplification was performed with a
real-time PCR system (ABI Prism 7500). The amplifica-
tion was performed as follows: 94°C for 15 min followed
by 50 cycles of 94°C for 15 s, 60°C for 60 s, and 72°C for
10 min with a real-time PCR system (ABI Prism 7500). The
data
are
expresses
as
a
relative
value
after
normalization to the GAPDH expression. Results The predominant observations of vehicle KK-Ay
mice glomeruli were diffuse mesangial matrix expansion
and capillary wall thickening (Figure 2A, G), which were
considered a hallmark of diabetic nephropathy. Moreover,
this mesangial matrix increase produces the formation of
Kimmelstiel-Wilson Nodules in diabetic renal glomerular
tuft (Figure 2D). In Figure 2A, segmental sclerosis was
present in some KK-Ay mice glomeruli compared with
normal mice. However, mesangial matrix expansion and
capillary thickness in KK-Ay mice were reduced by Qiwei
granules treatment (Figure 2B, E, H). The reduction of
mesangial matrix expansion and fibrosis significantly dif-
fered with vehicle KK-Ay mice (Figure 2J, K). To investi-
gate whether Qiwei granules affected the renal ultra
microstructure, glomerular podocytes were examined by
transmission electron microscopy. In Figure 3, the foot
processes of podocyte were fusion and the GBM became
thicker in KK-Ay vehicle mice compared with normal
mice. With the treatment of Qiwei granules, the podocyte
injury especially the foot process fusion was ameliorated. These results showed that Qiwei granules mitigated the Results Effect of Qiwei granules on metabolic and renal parameters
We measured body weight, fasting blood glucose (FBG)
at 12 weeks of age, and confirmed no significant differences
between vehicle KK-Ay mice and Qiwei granules treated Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 4 of 11 Table 1 Biochemical parameters in each mouse at 12 and 22 weeks of age
KK-Ay
KK-Ay + Qiwei granules
C57BL/6J normal
(n = 6)
(n = 5)
(n = 6)
Body weight Initial (g)
39.67 ± 2.66
39.6 ± 3.49
29.33 ± 1.70**
Body weight Final (g)
47 ± 3.79
44.2 ± 5.31
31.33 ± 0.81**
Initial FBG (mmol/L)
24.69 ± 4.8
22.28 ± 6.28
5.48 ± 0.79**
Final FBG(mmol/L)
16.75 ± 9.51
5.8 ± 3.26*
3.98 ± 0.63**
Kidney weight (g)
0.30 ± 0.04
0.26 ± 0.05
0.19 ± 0.02**
Scr (μmol/L)
44.46 ± 12.17
25.73 ± 7.96**
24.73 ± 6.88**
BUN (mmol/L)
6.25 ± 0.73
6.37 ± 1.38
7.75 ± 0.85
Date expressed as means ± SD, *P < 0.05 and **P < 0.01compared with KK-Ay vehicle. Table 1 Biochemical parameters in each mouse at 12 and 22 weeks of age Date expressed as means ± SD, *P < 0.05 and **P < 0.01compared with KK-Ay vehicle. KK-Ay mice (Table 1). After 10 weeks treatment, body
weight of vehicle KK-Ay mice remained a high level com-
pared with normal mice, but did not differ from KK-Ay
mice with Qiwei granules treatment. At the end of
10 weeks treatment, FBG in vehicle KK-Ay mice was
higher than normal mice, and Qiwei granules significantly
decreased the level of FBG in KK-Ay mice (Table 1). The
kidney weight levels in Qiwei granules mice tended to be
lower than those in vehicle KK-Ay mice, although the
change was not statistically significant. Moreover, Qiwei
granules treatment alleviated the levels of Scr of KK-Ay
mice close to normal mice compared with vehicle KK-Ay
mice. 24 h urinary protein in vehicle KK-Ay mice was
remarkablely increased, and Qiwei granules significantly
decreased 24 h urinary protein after 8 weeks treatment
(Figure 1). In a word, administration of Qiwei granules
treatment showed a positive effect on the FBS, Scr, and
24 h urinary albumin in KK-Ay mice. Under light microscope, the glomerular changes among
three group mice at 22 weeks of age were shown in
Figure 2. Effects of Qiwei granules on KK-Ay mice renal
morphology The renal structure of each group was observed under
light microscopy and transmission electron microscopy. Figure 1 24 h urinary protein after 0, 4, 8 weeks treatment were measured in mice. Data of the urinary protein are expressed as mean ± SEM
(n = 6). *P < 0.05, **P < 0.01 compared with KK-Ay vehicle group. Figure 1 24 h urinary protein after 0, 4, 8 weeks treatment were measured in mice. Data of the urinary protein are expressed as mean ± SEM
(n = 6). *P < 0.05, **P < 0.01 compared with KK-Ay vehicle group. Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 5 of 11 Figure 2 Renal histopathology was observed after 10 weeks treatment in mice under light microscopy. (A-C) HE stained glomeruli, ×400
magnification. (D-F) PAS stained renal slices, ×400 magnification. (G-I) Masson’s Trichrome stained renal sections, ×400 magnification. (A, D, G)
KK-Ay vehicle mice. (B, E, H), KK-Ay mice treated with Qiwei granules. (C, F, I) C57BL/6J normal mice. (J) Glomerular mesangial matrix expansion
and (K) fibrosis of three groups were determined semi-quantitatively by PAS and Masson’s Trichrome staining. Data were presented as means ± SEM. *P < 0.05; **P < 0.01 compared to KK-Ay vehicle group. Figure 2 Renal histopathology was observed after 10 weeks treatment in mice under light microscopy. (A-C) HE stained glomeruli, ×400
magnification. (D-F) PAS stained renal slices, ×400 magnification. (G-I) Masson’s Trichrome stained renal sections, ×400 magnification. (A, D, G)
KK-Ay vehicle mice. (B, E, H), KK-Ay mice treated with Qiwei granules. (C, F, I) C57BL/6J normal mice. (J) Glomerular mesangial matrix expansion
and (K) fibrosis of three groups were determined semi-quantitatively by PAS and Masson’s Trichrome staining. Data were presented as means ± SEM. *P < 0.05; **P < 0.01 compared to KK-Ay vehicle group. renal injury of KK-Ay mice both under light microscopy
and transmission electron microscopy. than normal mice, while Qiwei granules prevented the
cell apoptosis compared with KK-Ay mice in Figure 4A. WT-1 is a positive marker of podocyte in kidney. As
shown in Figure 4B, WT-1 positive cells were signifi-
cantly decreased in the glomeruli of KK-Ay mice com-
pared with normal mice. However, Qiwei granules
preserved WT-1 positive cells in KK-Ay mice. The aver-
age number of podocytes stained with WT-1 antibody Effect of Qiwei granules on apoptosis and podocyte
marker WT-1 in KK-Ay mice kidneys Many podocytes, mesangial cells, and endothelial cells
were positively labeled by TUNEL in mice kidney. The
level of TUNEL positive cells in KK-Ay mice was higher Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 6 of 11 Figure 3 Ultrastructural features of renal tissue were observed after 10 weeks treatment in three group mice under transmission
electron microscopy (bar 1 μm). (A) KK-Ay vehicle mice, (B) KK-Ay mice treated with Qiwei granules, (C) C57BL/6J normal mice. Figure 3 Ultrastructural features of renal tissue were observed after 10 weeks treatment in three group mice under transmission
electron microscopy (bar 1 μm). (A) KK-Ay vehicle mice, (B) KK-Ay mice treated with Qiwei granules, (C) C57BL/6J normal mice. Figure 3 Ultrastructural features of renal tissue were observed after 10 weeks treatment in three group mice under transmission
electron microscopy (bar 1 μm). (A) KK-Ay vehicle mice, (B) KK-Ay mice treated with Qiwei granules, (C) C57BL/6J normal mice. compared with normal mice (Figure 5C, D). In addition,
the protein expression of integrin α3 and integrin β1
also decreased in vehicle KK-Ay mice (Figure 6A, C, D). However, the decreases in mRNA and protein expression
of integrin α3 and integrin β1were obviously ameliorated
with Qiwei granules treatment in KK-Ay mice. These re-
sults suggested that Qiwei granules regulated the protein
and mRNA expression of integrin α3 and integrin β1 in
KK-Ay mice. per glomerulus in Qiwei granules treatment group was
higher than that in the vehicle group (Figure 4C). These
results indicated that Qiwei granules prevented apop-
tosis and preserved the podocyte in KK-Ay mice. Effect of Qiwei granules on pAkt /Akt and cleaved
caspase-3/caspase-3 in KK-Ay mice renal tissue caspase 3/caspase 3 in KK A mice renal tissue
It has been shown that PI3K/AKT pathway related with
SD molecules (SDs) is one key part of podocyte cell sur-
vival signaling to maintain podocyte functional integrity
[28]. Our study found that Qiwei granules prevented the
podocyte apoptosis and increase the expression of SDs,
so we would investigate whether Qiwei granules regu-
lated
the
phosphorylation
of
Akt
and
cleavage
of
caspase-3. As shown in Figure 7, the protein expression
of pAkt in vehicle KK-Ay mice was significantly lower
compared with normal mice, while the expression of
total Akt was no difference between vehicle diabetic
mice and normal mice. With Qiwei granules treatment,
the expression of pAkt/Akt in KK-Ay mice was upregu-
lated compared with vehicle KK-Ay mice. Further we ex-
amined the protein level of caspase-3 cleavage, the key
downstream effector of apoptosis. The cleaved caspase-3
expression of vehicle diabetic mice was obviously higher
than normal mice. Exposure to Qiwei granules resulted in
decrease of cleaved caspase-3 expression in KK-Ay mice
kindey. Thus, Qiwei granules improved the phosphoryl-
ation of Akt and inhibited cleavage of caspase-3. Regulation of Qiwei granules on expression of integrin α3
and integrin β1 in KK-Ay mice renal tissue Integrin α3β1 mediates the adhesion of podocytes to the
glomerular basement membrance (GBM) which contain
a transmembrane region and intracellular region. So we
investigated both the mRNA and protein expression of
integrin α3β1 in KK-Ay mice kidney. As reports shown,
there were significantly downregulation of integrin α3
and integrin β1 mRNA expression in vehicle KK-Ay mice Enhancement of Qiwei granules on expression levels of
nephrin and CD2AP in KK-Ay mice renal tissue Nephrin is a major transmembrane protein in the podo-
cyte slit diaphragm which relates to renal tissue damage
in DN [9]. As shown in Figure 5A, we found that
nephrin mRNA expression in the renal tissue of KK-Ay
mice was lower than normal mice. And the level of
nephrin mRNA in Qiwei granules treated KK-Ay mice
was significantly higher than vehicle KK-Ay mice. We
further examined the expression of nephrin protein in
three groups. As expected, the western blot analysis
demonstrated that nephrin protein expression was reduced
in vehicle treated diabetic mice (Figure 6A, B). Qiwei gran-
ules markedly alleviated the reduction of nephrin protein
expression in diabetic kidney (Figure 6A, B). CD2AP, an-
other transmembrane protein, plays a great role in main-
tenance of structure and function of podocyte. The protein
expression of CD2AP was decreased in vehicle KK-Ay mice,
and treatment with Qiwei granules ameliorated the de-
crease (Figure 6A, C). These data suggested Qiwei gran-
ules improved expression of nephrin and CD2AP protein
expression in KK-Ay mice renal podocytes. Discussion Due to the constant increase in the incidence of type 2
diabetes mellitus, diabetic nephropathy has become the Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 7 of 11 Figure 4 Photomicrograph of cell apoptosis and podocyte marker WT-1 in KK-Ay mice kidney. (A) The apoptotic cells were labeled with
TUNEL (×400) in mice kidney. (B) The podocytes were perfumed by immunohistochemistry staining of the renal tissues for WT-1 (×400). (C) The
average WT-1 positive podocytes per glomerulus were expressed as means ± SEM (n = 40). **P < 0.01 compared to KK-Ay vehicle group. Figure 4 Photomicrograph of cell apoptosis and podocyte marker WT-1 in KK-Ay mice kidney. (A) The apoptotic cells were labeled with
TUNEL (×400) in mice kidney. (B) The podocytes were perfumed by immunohistochemistry staining of the renal tissues for WT-1 (×400). (C) The
average WT-1 positive podocytes per glomerulus were expressed as means ± SEM (n = 40). **P < 0.01 compared to KK-Ay vehicle group. highly prevalent cause of chronic kidney disease world-
wide. In China traditional Chinese medicines have been
used for the diabetes mellitus and its complications for
thousand of years. Chinese medicines have been attracted
worldwide attention to become a new therapy for diabetic
nephropathy. Qiwei granule, one of such traditional
Chinese medicines, has been comprised of 6 Chinese
herbs. In clinic study, Qiwei granule was shown to
decrease blood glucose, improve hyperlipidemia, and
reduce the proteinuria of DN patients [23]. The
present study showed that Qiwei granules treatment significantly improved the metabolic parameters, alle-
viated the albuminuria, and protected renal structure
to prevent progression of DN in KK-Ay mice. In
addition, the mitigation of podocyte lesion with Qiwei
granules treatment was further demonstrated, follow-
ing by the increases of SD molecules expression and
likely activating Akt signaling pathway. The classical hallmarks of diabetic nephropathy include
glomerular hypertrophy, expansion of mesangial matrix
and thickness of GBM [29]. Recently mounting researches
reveal that podoctye injury becomes a major contributor Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 8 of 11 Figure 5 The mRNA expression levels of slit diaphragm molecules in KK-Ay mice renal tissue. The mRNA expression levels of (A) nephrin,
(B) integrin α3, and (C) integrin β1 were assessed by real-time PCR. The data were expressed as means ± SEM (n = 5) normalized to GAPDH
mRNA expression. Discussion Figure 7 Effect of Qiwei granules on phosphorylation of Akt and cleavage of caspase-3 signaling in the kidney of KK-Ay mice. (A) Protein
expression levels of pAkt, Akt, cleaved caspase-3 and caspase-3 in renal tissues of three groups were analyzed by western blotting. Rate of Akt
phosphorylation and caspase-3 activation were shown in (B) and (C). Data were presented as mean ± SEM (n = 5) normalized to GAPDH protein
expression. *P < 0.05; **P < 0.01 compared to KK-Ay vehicle group. Apoptosis, one of the considered causes of podocyte
loss results in the development of albuminuria in dia-
betic nephropathy. Akt is a serine/threonine protein
kinase that plays a critical role in cell survival by inhibit-
ing the pro-apoptotic signals. Previous studies show that
nephrin and CD2AP stimulate Akt signaling pathway
and thereby reduce podocyte apoptosis to protect podo-
cyte in glomerular [34,35]. Our results also showed that
the expression of nephrin and CD2AP was decreased,
while the phosphorylation of Akt was inhibited and in-
duced the apoptosis in diabetic KK-Ay mice. Administra-
tion of Qiwei granules improved the phosphorylation of
Akt and increased the expression of SDs contributing to
preservation of podocyte. Caspase-3 is a critical execu-
tioner of apoptosis and operates as the key effector en-
zyme in cell death. Activation of caspase-3 plays an
important role in apoptosis. In accordance with previ-
ous studies [36,37], our study showed that cleaved
caspase-3 expression was increased in renal tissue of
diabetic KK-Ay mice. Qiwei granules reduced the overex-
pression of cleaved caspase-3 in diabetic mice possibly
leading to inhibit apoptosis. These results indicated that
Qiwei granules maybe affect podocyte apoptosis depend-
ing on Akt pathway and caspase-3. granules both reversed the glomerular and podocyte
injuries. Slit diaphragm molecules between podocytes com-
posed of multiple protein complexes contribute to the
glomerular filtration barrier. Derangement of SDs re-
sults in proteinuria. Nephrin is a major transmembrane
protein located in the glomerular SD region. The altered
expression of nephrin has been observed in the initi-
ation of proteinuria and linked to the development of
proteinuria in DN [29,32]. CD2AP, a transmembrane
protein interacted with nephrin, preserves the functions
of cytoskeleton and SD. Damage to CD2AP leads to
disrupting podocyte cytoskeleton, and inducing massive
proteinuria [9]. Basement membrane-podocyte junctional
membrane proteins such as integrin α3β1 also play a vital
role in preserving the normal structure and functioning of
podocyte [13]. Discussion *P < 0.05; **P < 0.01 compared to KK-Ay vehicle group. Figure 5 The mRNA expression levels of slit diaphragm molecules in KK-Ay mice renal tissue. The mRNA expression levels of (A) nephrin,
(B) integrin α3, and (C) integrin β1 were assessed by real-time PCR. The data were expressed as means ± SEM (n = 5) normalized to GAPDH
mRNA expression. *P < 0.05; **P < 0.01 compared to KK-Ay vehicle group. barrier resulting in albuminuria [30,31]. In our study, the
mesangial matrix expansion and GBM thickening were
observed in KK-Ay mice. In addition, podocyte lesion
demonstrated as foot process effacement and podocyte
loss was found in KK-Ay mice. The treatment of Qiwei to the albuminuria in diabetic patients and animal models
[3,4]. It relates to the importance of podocyte in maintain-
ing the filtration barrier. However, the podocyte injuries
including foot process effacement, detachment, hyper-
trophy, and apoptosis lead to the dyfunction of filtration Figure 6 The protein expression levels of slit diaphragm molecules in the KK-Ay mice kidney. (A) Protein expression levels of nephrin,
CD2AP, integrin α3, and integrin β1 were analyzed by western blotting in renal tissues of three groups. (B-E) Data were presented as mean ± SEM
(n = 5) normalized to GAPDH protein expression. *P < 0.05; **P < 0.01 compared to KK-Ay vehicle group. Figure 6 The protein expression levels of slit diaphragm molecules in the KK-Ay mice kidney. (A) Protein expression levels of nephrin,
CD2AP, integrin α3, and integrin β1 were analyzed by western blotting in renal tissues of three groups. (B-E) Data were presented as mean ± SEM
(n = 5) normalized to GAPDH protein expression. *P < 0.05; **P < 0.01 compared to KK-Ay vehicle group. Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 9 of 11 Figure 7 Effect of Qiwei granules on phosphorylation of Akt and cleavage of caspase-3 signaling in the kidney of KK-Ay mice. (A) Protein
expression levels of pAkt, Akt, cleaved caspase-3 and caspase-3 in renal tissues of three groups were analyzed by western blotting. Rate of Akt
phosphorylation and caspase-3 activation were shown in (B) and (C). Data were presented as mean ± SEM (n = 5) normalized to GAPDH protein
expression. *P < 0.05; **P < 0.01 compared to KK-Ay vehicle group. Competing interests The authors declare that they have no competing interests. 15. Zhang J, Xie X, Li C, Fu P. Systematic review of the renal protective effect of
Astragalus membranaceus (root) on diabetic nephropathy in animal models. J Ethnopharmacol. 2009;126:189–96. 15. Zhang J, Xie X, Li C, Fu P. Systematic review of the renal protective effect of
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mice. Mol Cell Biol. 2004;24:550–60. Authors’ contributions JZ designed the study, conducted the experiments, and wrote the
manuscript. WS and HY performed the experiments, analyzed the data. LQ,
LL and LW participated in the animal experiments. XG and XW performed
the experiments and manuscript preparation. YZ drafted the manuscript. TH
and MG provided the original idea for the work. TL designed and instructed
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in the treatment of diabetic nephropathy in rats. Am J Chin Med. 2012;40:1177–87. 17. Shieh JP, Cheng KC, Chung HH, Kerh YF, Yeh CH, Cheng JT. Plasma glucose
lowering mechanisms of catalpol, an active principle from roots of
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vulgaris L. in streptozotocin-induced diabetic mice. Asia Pac J Clin Nutr. 2007;16 Suppl 1:427–31. Abbreviations
BUN Bl
d
i BUN: Blood urine nitrogen; BW: Body weight; CD2AP: CD2 associated protein;
DN: Diabetic nephropathy; ESRD: End-stage renal disease; FBG: Fasting blood
glucose; GBM: Glomerular basement membrane; HE: Hematoxylin and eosin;
PAS: Periodic acid-Schiff; Scr: Serum creatinine; SD: Slit diaphragm;
TUNEL: TdT-mediated dUTP nick end labeling; WT-1: Wilms tumor −1. 13. Kreidberg JA, Donovan MJ, Goldstein SL, Rennke H, Shepherd K, Jones RC,
et al. Alpha 3 beta 1 integrin has a crucial role in kidney and lung
organogenesis. Development. 1996;122:3537–47. 14. Fang J, Wei H, Sun Y, Zhang X, Liu W, Chang Q, et al. Regulation of
podocalyxin expression in the kidney of streptozotocin-induced diabetic rats
with Chinese herbs (Yishen capsule). BMC Complement Altern Med. 2013;13:76. Conclusions In conclusion, the Chinese formulation Qiwei granules
protects podocyte in the kidney of diabetic KK-Ay mice. Qiwei granules treatment significantly improved the ex-
pression of SD molecules nephrin, CD2AP, integrin α3β1. Furthermore, it appears to activate Akt signaling pathway
and inhibit the apoptosis in the kidney. It alleviated the
podocyte lesion and glomerular pathologic changes, which
resulted in the decrease of albuminuria and protection of
renal function. Our study suggests that Qiwei granules
ameliorates podocyte lesion to prevent progression of DN
in KK-Ay mice. 7. Schwarz K, Simons M, Reiser J, Saleem MA, Faul C, Kriz W, et al. Podocin, a
raft-associated component of the glomerular slit diaphragm, interacts with
CD2AP and nephrin. J Clin Invest. 2001;108:1621–9. 8. Jim B, Ghanta M, Qipo A, Fan Y, Chuang PY, Cohen HW, et al. Dysregulated
nephrin in diabetic nephropathy of type 2 diabetes: a cross sectional study. PLoS One. 2012;7:e36041. 8. Jim B, Ghanta M, Qipo A, Fan Y, Chuang PY, Cohen HW, et al. Dysregulated
nephrin in diabetic nephropathy of type 2 diabetes: a cross sectional study. PLoS One. 2012;7:e36041. 9. Toyoda M, Suzuki D, Umezono T, Uehara G, Maruyama M, Honma M, et al. Expression of human nephrin mRNA in diabetic nephropathy. Nephrol Dial
Transplant. 2004;19:380–5. 10. Benzing T. Signaling at the slit diaphragm. J Am Soc Nephro 10. Benzing T. Signaling at the slit diaphragm. J Am Soc Nephrol. 2004;15:1382–91. 11. Kagami S, Kondo S. Beta1-integrins and glomerular injury. J Med Invest. 2004;51:1–13. 11. Kagami S, Kondo S. Beta1-integrins and glomerular injury. J Med Invest. 2004;51:1–13. 12. Chen HC, Chen CA, Guh JY, Chang JM, Shin SJ, Lai YH. Altering expression
of alpha3beta1 integrin on podocytes of human and rats with diabetes. Life
Sci. 2000;67:2345–53. 12. Chen HC, Chen CA, Guh JY, Chang JM, Shin SJ, Lai YH. Altering expression
of alpha3beta1 integrin on podocytes of human and rats with diabetes. Life
Sci. 2000;67:2345–53. Acknowledgements Acknowledgements
This study was supported by grants from National Major Scientific and
Technological Special Project for Significant New Drugs
Development(2011ZX09101-004-04), Innovation Team Project of Beijing
University of Chinese Medicine (No.2011-CXTD-19), Ministry of Education Key
Laboratory of Health Preservation of Chinese Medicine and the key project
of Chinese Ministry of Education (No. 311011). This study was supported by grants from National Major Scientific and
Technological Special Project for Significant New Drugs
Development(2011ZX09101-004-04), Innovation Team Project of Beijing
University of Chinese Medicine (No.2011-CXTD-19), Ministry of Education Key
Laboratory of Health Preservation of Chinese Medicine and the key project
of Chinese Ministry of Education (No. 311011). 19. Valentova K, Truong NT, Moncion A, de Waziers I, Ulrichova J. Induction of
glucokinase mRNA by dietary phenolic compounds in rat liver cells in vitro. J Agric Food Chem. 2007;55:7726–31. 20. Hwang SM, Kim JS, Lee YJ, Yoon JJ, Lee SM, Kang DG, et al. Anti-diabetic
atherosclerosis effect of Prunella vulgaris in db/db mice with type 2
diabetes. Am J Chin Med. 2012;40:937–51. Discussion The downregulation of integrin α3β1 re-
ceptors detach podocytic processes from GBM and results
in proteinuria [33]. Our finding suggested that expressions
of nephrin, CD2AP, and integrin α3β1 was markedly de-
creased in diabetic KK-Ay mice compared with normal
mice, whereas Qiwei granules treatment restored the SDs
expressions. In combination, these results indicated that
Qiwei granules treatment increased the expression levels
of nephrin, CD2AP, and integrin α3β1 to protect from
podocyte injury in diabetic KK-Ay mice. Previous studies are reported that high glucose induces
podocyte apoptosis and reduction of SDs expressions Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Page 10 of 11 contributing to podocyte injury [38]. In the present
study, Qiwei granules decreased the blood glucose and
protected the podocyte lesion to prevent the progress of
DN. Qiwei granules consists of Astragalus membrana-
ceus, Euonymus alatus, Rehmannia glutinosa, Prunella
vulgaris, and Panax notoginseng that these herbs are
shown to decreases the glucose, and protect diabetic
nephropathy kidney [15-17,19,20]. In a word, Qiwei
granules alleviates podocyte lesion maybe not only pro-
tecting podocyte directly, but also related with reducing
blood glucose. This result requires further confirmation
in the future. Received: 22 July 2014 Accepted: 9 March 2015 Author details
1 21. Guang-Rong Z, Zhi-Jun X, Ting-Xiang Y. Antioxidant activities of Salvia
miltiorrhiza and Panax notoginseng. Food Chem. 2006;99:767–74. 1Beijing University of Chinese Medicine, 11 North 3rd-ring East Road, 100029
Chaoyang District, Beijing, People’s Republic of China. 2Dongzhimen Hospital
Eastern Affilated to Beijing University of Chinese Medicine, 116 Cuiping West
Road, 100029 Tongzhou District, Beijing, People’s Republic of China. 3School
of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Koshien
Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan. 22. Peng SL, Guo ZA. Effect of total saponins of Panax notoginseng on urinary
albumin in patients with chronic renal failure. Chinese Critical Care
Medicine. 2010;22:744–6. 23. Jinxi Z, Xin M, Shidong W. Therapeutic Observations on Renal Insufficiency
of Diabetic Nephropathy in Its Compensatory Phase Treated by Zhixiao
Tongmai Ning Granule: A Report of 33 Cases. Henan Traditional Chinese
Medicine. 2004;24:20–2. 23. Jinxi Z, Xin M, Shidong W. Therapeutic Observations on Renal Insufficiency
of Diabetic Nephropathy in Its Compensatory Phase Treated by Zhixiao
Tongmai Ning Granule: A Report of 33 Cases. Henan Traditional Chinese
Medicine. 2004;24:20–2. Received: 22 July 2014 Accepted: 9 March 2015 Page 11 of 11 Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 24. Minzhou L, Yanbin G, Mingfei M. The effect of Qiwei granules on TGF-β1/
Smads signaling pathway in KK-Ay mice. J Tradit Chin Med. 2013;54:1141–4. 24. Minzhou L, Yanbin G, Mingfei M. The effect of Qiwei granules on TGF-β1/
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KK mice. Endocrinol Jpn. 1970;17:23–35. 26. Castle CK, Colca JR, Melchior GW. Lipoprotein profile characterization of the
KKA(y) mouse, a rodent model of type II diabetes, before and after
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nephropathy in KK and KK-Ay mice. Exp Anim. 2002;51:191–6. 8. Xavier S, Niranjan T, Krick S, Zhang T, Ju W, Shaw AS, et al. TbetaRI inde 28. Xavier S, Niranjan T, Krick S, Zhang T, Ju W, Shaw AS, et al. TbetaRI independently
activates Smad- and CD2AP-dependent pathways in podocytes. J Am Soc
Nephrol. 2009;20:2127–37. 29. Zhou et al. BMC Complementary and Alternative Medicine (2015) 15:97 Author details
1 Jefferson JA, Shankland SJ, Pichler RH. Proteinuria in diabetic kidney disease:
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https://openalex.org/W2743592042
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https://repositorio.unesp.br/bitstream/11449/159675/1/WOS000408224400001.pdf
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English
| null |
Coordinated Role of Toll-Like Receptor-3 and Retinoic Acid-Inducible Gene-I in the Innate Response of Bovine Endometrial Cells to Virus
|
Frontiers in immunology
| 2,017
|
cc-by
| 8,688
|
Original Research
published: 23 August 2017
doi: 10.3389/fimmu.2017.00996 Keywords: endometrium, pattern-recognition receptor, toll-like receptor, virus, cytokine, chemokine, poly(I:C),
RIG-I Coordinated Role of Toll-Like
Receptor-3 and Retinoic
Acid-Inducible Gene-I in the Innate
Response of Bovine Endometrial
Cells to Virus Luisa C. Carneiro1,2, Carmen Bedford1, Sarah Jacca1,3, Alfonso Rosamilia1,3,
Vera F. de Lima2, Gaetano Donofrio3, I. Martin Sheldon1 and James G. Cronin1* 1 Institute of Life Science, Swansea University Medical School, Swansea, United Kingdom, 2 Faculty of Agricultural and
Veterinary Science, Universidade Estadual Paulista, Jaboticabal, Brazil, 3 Department of Medical-Veterinary Science,
University of Parma, Parma, Italy Bovine herpesvirus-4 (BoHV-4) and bovine viral diarrhea virus (BVDV) infect the uterus
of cattle, often resulting in reduced fertility, or abortion of the fetus, respectively. Here,
exposure of primary bovine endometrial cells to BoHV-4 or BVDV modulated the
production of inflammatory mediators. Viral pathogen-associated molecular patterns
(PAMPs) are detected via pattern-recognition receptors (PRRs). However, the relative
contribution of specific PRRs to innate immunity, during viral infection of the uterus, is
unclear. Endometrial epithelial and stromal cells constitutively express the PRR Toll-like
receptor (TLR)-3, but, the status of retinoic acid-inducible gene I (RIG-I), a sensor of
cytosolic nucleic acids, is unknown. Primary endometrial epithelial and stromal cells had
low expression of RIG-I, which was increased in stromal cells after 12 h transfection with
the TLR3 ligand Poly(I:C), a synthetic analog of double-stranded RNA. Furthermore,
short interfering RNA targeting TLR3, or interferon (IFN) regulatory transcription factor 3,
an inducer of type I IFN transcription, reduced Poly(I:C)-induced RIG-I protein expression
and reduced inflammatory mediator secretion from stromal cells. We conclude that
antiviral defense of endometrial stromal cells requires coordinated recognition of PAMPs,
initially via TLR3 and later via inducible RIG-I. Edited by:
Wenzhe Ho,
Temple University School of
Medicine, United States
Reviewed by:
Yu Zhou,
Codiak Biosciences, United States
Haitao Guo,
University of North Carolina
at Chapel Hill, United States
*Correspondence:
James G. Cronin
j.cronin@swansea.ac.uk Edited by:
Wenzhe Ho,
Temple University School of
Medicine, United States Reviewed by:
Yu Zhou,
Codiak Biosciences, United States
Haitao Guo,
University of North Carolina
at Chapel Hill, United States Specialty section:
This article was submitted to
Microbial Immunology,
a section of the journal
Frontiers in Immunology Received: 24 February 2017
Accepted: 04 August 2017
Published: 23 August 2017 INTRODUCTION Carneiro LC, Bedford C, Jacca S,
Rosamilia A, de Lima VF, Donofrio G,
Sheldon IM and Cronin JG (2017)
Coordinated Role of Toll-Like
Receptor-3 and Retinoic
Acid-Inducible Gene-I in the Innate
Response of Bovine Endometrial
Cells to Virus. Front. Immunol. 8:996. doi: 10.3389/fimmu.2017.00996 The initiation of the innate immune response to viruses depends on the detection of pathogen-
associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) (1). The main fami
lies of PRRs are the Toll-like receptors (TLRs), nucleotide oligomerization domain-like receptors,
retinoic acid-inducible gene I (RIG-I)-like receptors, and C-type lectin receptors. These receptors
are most often expressed by hematopoietic cells, but the epithelial and stromal cells of the endo
metrium also possess functional PRRs. The PRRs that detect uterine bacteria that contaminate the
uterus, causing infertility, are well documented in cattle (2–9). However, viral infections also result August 2017 | Volume 8 | Article 996 Frontiers in Immunology | www.frontiersin.org 1 TLR3 RIG-1 Endometrium Carneiro et al. in decreased conception rates and abortion in cattle. Although,
little is known about the functional response of endometrial cells
in the detection and response to viruses or their PAMPs.h herpesvirus DNA and RIG-I, indicating a non-redundant role for
the sensing of herpesvirus by fibroblasts (31, 32). Furthermore,
BVDV induces expression of TLR3 and DDX58 (RIG-I) genes in
bovine endometrial cells (8). The double-stranded (ds)DNA Gammaherpesvirus bovine
herpesvirus-4 (BoHV-4) and the single-stranded (ss)RNA
Pestivirus bovine viral diarrhea virus (BVDV) infect the uterus
of cattle (10, 11). Gammaherpesviruses are ubiquitous pathogens
of animals and humans, and primary infections are usually
subclinical. However, BoHV-4 and BVDV can cause disease, par
ticularly if the host becomes immunologically or metabolically
stressed, a common occurrence in postpartum dairy cattle (12). BoHV-4 is tropic for endometrial epithelial and stromal cells,
and macrophages become persistently infected with virus (13). Increased replication of BoHV-4 also occurs in the endometrium
particularly following postpartum infection of the uterus with
the Gram-negative Escherichia coli, resulting in uterine disease
(14). Lipopolysaccharide (LPS), from the Gram-negative cell
wall, induces inflammatory cytokine secretion from endometrial
cells via TLR4 (3, 15). Another commonly isolated virus BVDV
can result in abortion of the fetus or in the birth of a persistently
infected calf, depending on the stage of gestation, and the devel
opment of the fetal immune system (10). INTRODUCTION In the present study, we investigated the innate immune
response of the endometrium to BVDV, BoHV-4, and to
representative PAMPs of the virus life cycle. In order to under
stand the recognition of viruses by endometrial epithelial and
stromal cells and the induction of pro-inflammatory mediators
and IFNs, we investigated the role of TLR3 and RIG-I in the
innate immune response to viable BoHV-4 and BVDV, and viral
PAMPs. We show that endometrial stromal cells responded
to viable viruses by inducing inflammatory cytokines and
chemokines. However, epithelial and stromal cells did not
directly respond to transfected DNA or 5’-triphosphorylated
ssRNA, but initiate an innate immune response to dsRNA,
which was TLR3 dependent. Although, unchallenged stromal
cells did not express RIG-I. RIG-I levels increased following
dsRNA Poly(I:C) transfection, and this was dependent on TLR3. Therefore, the endometrial innate immune response required a
co-ordinated response to virus, involving TLR3 mediated pro-
inflammatory cytokine and chemokine expression, and dsRNA
induced RIG-I expression. Understanding the mechanisms of
innate immune activation of endometrial cells, and the etiol
ogy and pathogenesis of disease, may allow the development of
efficient antiviral strategies. The innate immune system is the first line of resistance to
viral infections, and plays a pivotal role in both the host’s early
response to viruses and subsequent adaptive immunity (16, 17). During viral infections, endosomal TLRs, and cytoplasmic RNA
helicases, including RIG-I, detect PAMPs, such as nucleic acids,
and initiate antiviral immunity via the induction of inflammatory
mediators and interferons (IFNs) (1). DNA viruses are usually
sensed via the DNA-dependent activator of interferon regula
tory factor (IRF) (DAI) that senses B-form DNA and induces
type I IFNs (18). However, DAI is not present in all cell types and
studies in DAI-deficient mice have failed to identify essential
roles for DAI in an innate antiviral response to herpesviruses
(19). Therefore, DAI is not thought to be the main PRR in innate
defense against herpesviruses. Viral DNA is also sensed by cyclic
GMP-AMP synthase (cGAS) and gamma-interferon-inducible
protein, which depend on the adaptor protein stimulator of
IFN genes (STING) (20, 21). However, DNA viruses can also
be sensed indirectly by the endosomal TLR3 after translation
of dsRNA during viral replication (22). dsRNA is part of the
life-cycle of all viruses, except negative-stranded RNA viruses
(23). Activation of TLR3, through binding of dsRNA, utilizes a
MyD88-independent pathway, via the TIR-domain-containing
adapter-inducing IFN-β (TRIF). INTRODUCTION Activation of this pathway
leads to phosphorylation of nuclear-factor κB (NF-κB), which
stimulates inflammatory cytokine gene expression. Alternatively,
TRIF signaling can activate TANK binding-kinase-1 (TBK-1)
and IRF-3, which is a key transcription factor responsible for
type I IFN gene expression (24, 25). Frontiers in Immunology | www.frontiersin.org Endometrial Cell Culture Uteri with no gross evidence of genital disease or microbial infec
tion were collected from cattle processed as part of the normal
work of an abattoir, as described previously (3). Endometrial epi
thelial and stromal cell populations were isolated, and the absence
of immune cell contamination confirmed by cell morphology
and by FACS analysis, as described previously (2). The cells
were plated at a density of 1 × 105 cells/ml in 6-, 12- or 24-well
plates (TPP, Trasadingen, Switzerland) in complete medium:
RPMI 1640 (Gibco, Life Technologies, UK), supplemented
with 10% heat-inactivated, endotoxin-free, fetal bovine serum
(BioSera, East Sussex, UK), 50 IU/ml penicillin (Sigma-Aldrich
Ltd., Dorset, UK), 50 µg/ml streptomycin (Sigma-Aldrich Ltd.),
2.5 µg/ml amphotericin B (Sigma-Aldrich Ltd.), and maintained
in a humidified, 5% CO2 in air atmosphere incubator at 37°C. i
Endometrial tissue for organ culture was collected from the
intercaruncular areas of the endometrium, using sterile 8-mm
diameter biopsy punches (Stiefel Laboratories Ltd., High Wycome,
UK), as described previously (33). Tissues were cultured in 24-well
plates (TPP, Trasadingen, Switzerland) containing 2 ml complete
medium/well. The ex vivo organ cultures (EVOCs) were exposed
to LPS (1 µg/ml), viable BoHV-4 (1 × 106/ml virus particles) or
BVDV (1 × 106/ml virus particles) within 4 h of slaughter and
maintained in a humidified, 5% CO2 in air atmosphere incubator
at 37°C. Subsequently, supernatants were collected after 24 or
72 h for analysis of inflammatory mediators by enzyme-linked
immunosorbent assay (ELISA). Viral DNA is also transcribed to uncapped 5’-triphosphate
ssRNA by RNA polymerase III, which is sensed by cytosolic
RIG-I (16, 26–28). Pestivirus, such as BVDV, and Hepacivirus
are the only mammalian viruses that include an uncapped
5’-triphosphate ssRNA stage in their replication cycles (26, 29). Dendritic cells (DCs) recognize Herpes simplex virus-2 via TLR9
(30). However, studies have also shown a direct interaction of August 2017 | Volume 8 | Article 996 Frontiers in Immunology | www.frontiersin.org 2 TLR3 RIG-1 Endometrium Carneiro et al. indicated in Section “Results.” Prior to challenge, PAMPs were
diluted in OPTI-MEM media (Gibco, UK), containing DOTAP
(N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium
methyl-sulfate) liposomal transfection reagent (Sigma-Aldrich
Ltd.), and incubated for 5 min at room temperature. The
DOTAP/PAMP solution was then added to 1 ml of complete
media in a well of a 24-well plate, so that the final concentra
tion of DOTAP was 10 µg/ml. Each experiment was performed
using cells isolated from at least three independent animals. Immunoblotting Total cell lysate proteins were quantified using the DC Assay
(Bio-Rad, UK) and separated (10 μg/lane) using Laemmli buffer
(Sigma-Aldrich) and 10% (vol/vol) SDS-PAGE, as described
previously (15). Pre-stained molecular weight markers were run
in parallel lanes (Bio-Rad, UK). After electrophoresis, proteins
were transferred to a polyvinylidene difloride membrane (GE
Healthcare); non-specific sites were blocked using a solution
of 5% (wt/vol) BSA (Sigma-Aldrich) in Tris-buffered saline
(TBS; Sigma-Aldrich) for 1 h at 37°C with gentle agitation. Membranes were probed with antibodies targeting RIG-I
(#4200, Cell Signaling, Danvers, MA, USA); phosphorylated
p65 (Serine536; #3033, Cell Signaling); total p65 (#3987, Cell
Signaling); and phosphorylated TBK1 (serine172; #5483, Cell
Signaling). Protein loading was evaluated by examining β-actin
protein levels using a β-actin antibody (ab8226, Abcam, UK). Primary antibodies were used at 1:1,000 dilutions in 5% (wt/
vol) BSA, TBS, and 0.1% Tween 20 (pH 7.6; Sigma) overnight
at 4°C with gentle agitation. After incubation, membranes were
washed for 5× 5 min in TBS and 0.1% Tween 20. Membranes
were then incubated in secondary horseradish peroxidase-
conjugated antibody (Cell Signaling) in TBS and 0.1% Tween 20
for 1.5 h, and washed for 5× 5 min in TBS and 0.1% Tween 20
(pH 7.6). Steady-state levels of immunoreactive proteins were
visualized using enhanced chemiluminescence (Western C, Bio-
Rad). Densitometry of non-saturated signals was performed on
independent immunoblots, using the Quantity-one software
(Bio-Rad). Endometrial Cell Culture Treatments
Viruses Bovine herpesvirus-4-4EGFPΔTK and the NADL strain of
BVDV (34) were propagated by infecting confluent monolayers
of Madin–Darby bovine kidney cells (MDBK) at a multiplicity of
infection (m.o.i.) of 0.5 tissue cell infectious doses/50 (TCID50)
per cell and maintained in minimal essential media (MEM;
Gibco, UK) with 2% FBS for 2 h. The medium was then removed
and replaced with fresh MEM containing 10% FBS. The virus was
purified when 90% of the cell monolayer exhibited a cytopathic
effect (CPE), at approximately 72 h post-infection. Cell-associated
virions were freed by three cycles of freeze-thawing at −80°C. Cell
debris was removed by centrifugation, and virions were pelleted
through a 3 ml cushion of 30% sucrose in PBS, in a Beckman 70
Ti rotor at 35,000 rpm for 90 min at 4°C. Viral pellets were resus
pended in cold MEM without FBS and TCID50 were determined
on MDBK cells by serial dilutions (35). Bovine endometrial stromal or epithelial cells were challenged
with BoHV-4 or BVDV at 1 m.o.i.. Explants were challenged with
1 × 106/ml virus particles. The supernatants were harvested and
analyzed by ELISA. Endometrial Cell Culture Supernatants were collected and stored at −20°C, while cells
were washed, and cell lysates collected using PhosphoSafe™
Extraction Reagent (EMD Millipore, UK) and stored at −80°C
until further processing for immunoblotting. Enzyme-Linked Immunosorbent Assay Enzyme-Linked Immunosorbent Assay
Concentrations of bovine IL-6 or IL-1β were measured by ELISA
according to the manufacturer’s instructions (Bovine IL-6 ELISA
Reagent Kit ESS0029; Bovine IL-1 beta ELISA Reagent Kit
ESS0027; Thermo Scientific, Cramlington, UK). Bovine IL-8 was
measured by ELISA, as described previously (36). Monocyte-Derived DCsi y
Forty-five milliliters of bovine blood, collected from the vena
jugularis externa, were immediately transferred into a tube
containing 5 ml sodium citrate (3.2%; Sigma-Aldrich) at the
local abattoir. In the laboratory, the blood was mixed with an
equal volume of Dulbecco’s phosphate buffered saline (D-PBS;
Sigma-Aldrich) and 16 ml of this diluted blood sample was
carefully layered onto 12 ml Ficoll-Paque PLUS (1.084 g/ml; GE
Healthcare, UK) in a 50 ml centrifuge tube (Falcon, UK), which
was centrifuged for 40 min at 400 × g. The mononuclear layer was
carefully collected from the interface, between the red-blood cells
and lymphocytes, and transferred to a sterile 15 ml centrifuge
tube (Falcon, UK) and D-PBS was added to the tube to a final
volume of 14 ml. The cells were resuspended by gentle pipetting
and the tube was centrifuged at 400 × g for 15 min. The super
natant was removed and the mononuclear cells resuspended in
14 ml of D-PBS. After centrifugation at 400 × g for 10 min, cells
were resuspended in 3 ml serum-free RPMI 1640 and transferred
to a 6 cm diameter Petri dish (NUNC, UK). After 30 min, the
cells were washed in 6 ml D-PBS and cultured in 3 ml complete
medium supplemented with IL-4 (25 ng/ml; Kingfisher Biotech,
St. Paul, MN, USA) and GM-CSF (25 ng/ml; Kingfisher Biotech)
to induce differentiation into DCs. Media was half-changed every
3 days, to avoid disturbing the cells, and the cells were cultured
for 9 days before treatment. RESULTS To characterize the innate immune response of the endometrium
to viruses, endometrial EVOCs, endometrial epithelial cells, and
endometrial stromal cells were exposed to BoHV-4 or BVDV
virus for 24 or 72 h. The prototypical PAMP, LPS, was used as
a positive control, since LPS induces inflammatory cytokine
secretion from endometrial cells (3, 15, 33). Indeed, EVOC
supernatants accumulated IL-6, IL-8, and IL-1β (Figures 1A–C),
and epithelial (Figures 1D,E) and stromal cells (Figures 1F,G)
accumulated IL-6 and IL-8 in response to LPS. Bovine herpesvirus-4 did not induce cytokine production in
EVOCs, whereas BVDV induced production of IL-6, IL-8, and
IL-1β (Figures 1A–C). BoHV-4 and BVDV resulted in reduced
accumulation of IL-6, but increased IL-8 accumulation from
epithelial cells, when compared with control (Figures 1D,E). Whereas, supernatants of stromal cells accumulated IL-6
after exposure to BVDV and IL-8 on exposure to BoHV-4 Short Interfering RNA (siRNA) Ninety per cent confluent endometrial epithelial cells, stromal
cells, or monocyte-derived DCs were challenged with the follow
ing PAMPs: dsDNA CpG dsDNA (ODN 2007), ssRNA (ssPolyU
Naked), dsRNA Poly(I:C) low-molecular weight (LMW),
dsRNA Poly(I:C) high-molecular weight (HMW), with Lipid A
or ultrapure LPS from E. coli 0111:B4 as a positive control (all
InvivoGen, Toulouse, France) for the times and concentrations g
(
)
Primary endometrial epithelial and stromal cells were trans
fected using Lipofectamine RNAiMAX Reagent (Invitrogen)
and siRNA (designed using Dharmacon’s siDESIGN Center,
Thermo Fisher Scientific, UK) targeting TLR3, IRF3, TRAF3,
or MYD88 (duplex sequences in Supplemental Table 1), as
described previously (15). Briefly, RNAiMAX–RNAi duplex August 2017 | Volume 8 | Article 996 Frontiers in Immunology | www.frontiersin.org 3 Carneiro et al. TLR3 RIG-1 Endometrium complexes were formed by adding 50 pM of siRNA to 500 µl of
Opti-MEM I Reduced Serum Media (Invitrogen) in each well of
a 6-well plate, with 50 pM of ON-TARGETplus Non-targeting
siRNA #1 (Dharmacon) as a control. Then, 7.5 µl RNAiMAX was
added to each well containing the diluted RNAi molecules and
left for 20 min at room temperature. Exponentially growing cells
(7 × 105 epithelial cells, 5 × 105 stromal cells) were then seeded
in 2.5 ml of RPMI 1640 growth media, supplemented with 10%
FBS, per well to give approximately 50% confluency. Poly(I:C)
(1 µg/ml) challenge was carried out 48 h after siRNA treatment
of cells, and cells and supernatants collected after a further 24 h,
for immunoblotting and ELISA experiments. Viable BoHV-4 or
BVDV challenge (1 m.o.i.) was carried out 48 h after siRNA treat
ment of cells, and cells and supernatants collected after a further
72 h, for immunoblotting and ELISA experiments. stated in Section “Results,” using SPSS 16.0 (SPSS Inc.). Values of
P < 0.05 were designated as significant. stated in Section “Results,” using SPSS 16.0 (SPSS Inc.). Values of
P < 0.05 were designated as significant. Statistical Analysis Endometrial cells were isolated independently, with the animal
designated as the statistical unit. Data are presented as mean
+ SEM and treatments were compared by two-way analysis of
variance with Dunnett’s post-comparison test, unless otherwise FIGURE 1 | Viable bovine herpesvirus-4 (BoHV-4) and bovine viral diarrhea virus (BVDV) induce inflammatory responses in endometrial tissue and cells. Ex vivo
organ cultures [EVOCs; (A–C); n = 6 independent animals], epithelial cells [(D,E); n = 4], or stromal cells [(F,G); n = 4] were exposed to lipopolysaccharide (1 µg/ml),
BoHV-4 [cells at 1 multiplicity of infection (m.o.i.); explants at 1 × 106 viral particles/ml], BVDV (cells at 1 m.o.i.; explants at 1 × 106 viral particles/ml) for 24 or 72 h. Concentrations of IL-6 (A,D,F), IL-8 (B,E,G), and IL-1β (C) in supernatants were measured by enzyme-linked immunosorbent assay. Data are presented as
mean + SEM, and analyzed by analysis of variance, using the post hoc Dunnett’s multiple comparison test to compare treatments with control, ***P < 0.001,
**P < 0.01, *P < 0.05. FIGURE 1 | Viable bovine herpesvirus-4 (BoHV-4) and bovine viral diarrhea virus (BVDV) induce inflammatory responses in endometrial tissue and cells. Ex vivo
organ cultures [EVOCs; (A–C); n = 6 independent animals], epithelial cells [(D,E); n = 4], or stromal cells [(F,G); n = 4] were exposed to lipopolysaccharide (1 µg/ml),
BoHV-4 [cells at 1 multiplicity of infection (m.o.i.); explants at 1 × 106 viral particles/ml], BVDV (cells at 1 m.o.i.; explants at 1 × 106 viral particles/ml) for 24 or 72 h. Concentrations of IL-6 (A,D,F), IL-8 (B,E,G), and IL-1β (C) in supernatants were measured by enzyme-linked immunosorbent assay. Data are presented as
mean + SEM, and analyzed by analysis of variance, using the post hoc Dunnett’s multiple comparison test to compare treatments with control, ***P < 0.001,
**P < 0.01, *P < 0.05. August 2017 | Volume 8 | Article 996 Frontiers in Immunology | www.frontiersin.org TLR3 RIG-1 Endometrium Carneiro et al. endometrial epithelial and stromal cells. BoHV-4 is a dsDNA
virus, and TLR9 detects unmethylated CpG dinucleotides in
viral DNA, inducing innate immune responses via the adaptor
MyD88 (30, 37). Here, transfected unmethylated CpG dsDNA
(ODN 2007) did not significantly induce accumulation of IL-6
(Figures 2A,C) or IL-8 (Figures 2B,D) from endometrial
epithelial or stromal cells. Uterine DCs have been implicated in
pregnancy maintenance and serve as antigen-presenting cells (Figures 1F,G). Statistical Analysis DCs
recognize Herpes simplex virus-2 via TLR9 (30). So, next we
investigated the response of blood-derived DCs to transfected
unmethylated CpG dsDNA (ODN 2007). DC supernatants did
not accumulate IL-6 or IL-8 in response to transfected ODN
2007 (Figures 2E,F). TLR3 and DDX58 genes (5, 8). Thus, we investigated RIG-I status
in bovine endometrial cells in response to Poly(I:C). RIG-I was
barely detectable in untreated cells (Figures 4A,B), but RIG-I
was induced in a time- and concentration-dependent manner in
endometrial stromal cells (Figures 4B,C). The activation dynamics of TLR3 by Poly(I:C) are influenced
by various factors, including size of the ligands. Therefore, we
tested the effects of transfecting LMW or HMW Poly(I:C) on key
innate immune signaling molecules initiated by viral infections,
using the previously tested PAMPs as comparators. Neither
DNA or ssRNA induced RIG-I expression, or phosphorylation
of NF-kB (serine536) or TBK1 (serine172), key innate immune
signaling intermediates in antiviral immunity (Figures 5A–C). Whereas, both transfected LMW and HMW Poly(I:C) induced
expression of RIG-I, phosphorylation of NF-kB (serine536), and
phosphorylation of TBK1 (serine172) in endometrial stromal
cells (Figures 5A–C). Bovine viral diarrhea virus is a positive ssRNA virus. Endosomal TLR7 and TLR8 recognize ssRNA (39, 40). Here,
transfected ssRNA did not induce increased IL-6 or IL-8 in
epithelial or stromal cell supernatants (Figures 2A–D). However,
DCs did accumulate IL-6 and IL-8 in response to transfected
ssRNA (Figures 2E,F). g
As a PAMP, dsRNA is an important activator of innate
immunity against viral infection (1). Here, Poly(I:C), which
mimics dsRNA, did not induce increased accumulation of IL-6
or IL-8 from epithelial cells or DCs (Figures 2A,B,E,F). Whereas,
Poly(I:C) induced IL-6 and IL-8 production from stromal cells
(Figures 2C,D). With stromal cells, Poly(I:C) did not induce
IL-6 accumulation in cell supernatants without the transfection
reagent DOTAP. This indicates that the induction of IL-6 by
Poly(I:C) requires initiation of intracellular sensing pathways
(Figure 2C). Furthermore, Poly(I:C) induced a time-dependent
increase in IL-6 and IL-8 accumulation from stromal cells, but
not epithelial cells (Figures 3A–D). This indicates that Poly(I:C)
activates pathways that lead to proinflammatory production in
stromal cells, but not epithelial or DCs. g
As NF-kB and TBK1 signal downstream of activated TLR3
(24, 25), we next investigated TLR3 and related signaling mol
ecules for their influence on RIG-I expression, and IL-6 and
IL-8 accumulation in endometrial stromal cell supernatants. Statistical Analysis Taken together, these data provide evidence that
endometrial tissue and cells sense and respond to viruses that
commonly induce uterine disease, by modulating inflammatory
mediator production. However, this response seems to depend on
the virus and the cell type of the endometrium. yp
Next, we explored whether transfection of PAMPs,
analogous to those produced at different stages of virus life-
cycle, induced production of cytokines, or chemokines from FIGURE 2 | Double-stranded RNA (dsRNA) induces inflammatory mediator production in endometrial cells and dendritic cells (DCs). Epithelial cells [(A,B); n = 7
independent animals], stromal cells [(C,D); n = 5], or DCs [(E,F); n = 4] were exposed to (white bars) or transfected (+DOTAP; black bars) with Lipid A (0.1 µg/ml),
CpG DNA (1 µg/ml), ssRNA (1 µg/ml), dsRNA Poly(I:C) (low-molecular weight; 1 µg/ml), or Poly(I:C) (high-molecular weight; 1 µg/ml) for 24 h. Concentrations of IL-6
(A,C,E) and IL-8 (B,D,F) in supernatants were measured by enzyme-linked immunosorbent assay. Data are presented as mean + SEM, and analyzed by two-way
analysis of variance, using the post hoc Dunnett’s multiple comparison test to compare treatment with control, ***P < 0.001, **P < 0.01, *P < 0.05. FIGURE 2 | Double-stranded RNA (dsRNA) induces inflammatory mediator production in endometrial cells and dendritic cells (DCs). Epithelial cells [(A,B); n = 7
independent animals], stromal cells [(C,D); n = 5], or DCs [(E,F); n = 4] were exposed to (white bars) or transfected (+DOTAP; black bars) with Lipid A (0.1 µg/ml),
CpG DNA (1 µg/ml), ssRNA (1 µg/ml), dsRNA Poly(I:C) (low-molecular weight; 1 µg/ml), or Poly(I:C) (high-molecular weight; 1 µg/ml) for 24 h. Concentrations of IL-6
(A,C,E) and IL-8 (B,D,F) in supernatants were measured by enzyme-linked immunosorbent assay. Data are presented as mean + SEM, and analyzed by two-way
analysis of variance, using the post hoc Dunnett’s multiple comparison test to compare treatment with control, ***P < 0.001, **P < 0.01, *P < 0.05. Frontiers in Immunology | www.frontiersin.org August 2017 | Volume 8 | Article 996 5 TLR3 RIG-1 Endometrium Carneiro et al. with the ability to induce primary immune responses (38). DCs
recognize Herpes simplex virus-2 via TLR9 (30). So, next we
investigated the response of blood-derived DCs to transfected
unmethylated CpG dsDNA (ODN 2007). DC supernatants did
not accumulate IL-6 or IL-8 in response to transfected ODN
2007 (Figures 2E,F). with the ability to induce primary immune responses (38). Statistical Analysis FIGURE 4 | Double-stranded RNA (dsRNA) induces retinoic acid-inducible gene I (RIG-I) expression in endometrial stromal cells in a time- and
concentration-dependent manner. Epithelial (A) or stromal cells (B) were transfected with dsRNA Poly(I:C) (1 µg/ml) for 2–72 h (A,B) or in increasing concentrations
for 24 h (C). Total cell proteins were extracted, and analyzed by immunoblotting for RIG-I. Average peak densities of RIG-I were normalized to β-actin and are
presented as mean + SEM. Immunoblots are representative of two independent animal experiments. FIGURE 5 | Double-stranded RNA (dsRNA) induces retinoic acid-inducible gene I (RIG-I) expression and phosphorylation of p65 and TANK binding-kinase-1 (TBK1)
in endometrial stromal cells. Stromal cells (A,B,C) were transfected with Lipid A (0.1 µg/ml), CpG DNA (1 µg/ml), ssRNA (1 µg/ml), dsRNA Poly(I:C) (low-molecular
weight; 1 µg/ml), or Poly(I:C) (high-molecular weight; 1 µg/ml) for 24 h. Total cell proteins were extracted, and analyzed by immunoblotting for RIG-I (A), p65 or
phosphorylated p65 (S536) (B), and phosphorylated TBK1 (S172) (C). Average peak densities of RIG-I, phosphorylated p65, or phosphorylated TBK1 proteins were
normalized to β-actin and are presented as mean + SEM. Immunoblots are representative of two independent animal experiments. FIGURE 4 | Double-stranded RNA (dsRNA) induces retinoic acid-inducible gene I (RIG-I) expression in endometrial stromal cells in a time- and
concentration-dependent manner. Epithelial (A) or stromal cells (B) were transfected with dsRNA Poly(I:C) (1 µg/ml) for 2–72 h (A,B) or in increasing concentrations
for 24 h (C). Total cell proteins were extracted, and analyzed by immunoblotting for RIG-I. Average peak densities of RIG-I were normalized to β-actin and are
presented as mean + SEM. Immunoblots are representative of two independent animal experiments. FIGURE 4 | Double-stranded RNA (dsRNA) induces retinoic acid-inducible gene I (RIG-I) expression in endometrial stromal cells in a time- and
concentration-dependent manner. Epithelial (A) or stromal cells (B) were transfected with dsRNA Poly(I:C) (1 µg/ml) for 2–72 h (A,B) or in increasing concentrations
for 24 h (C). Total cell proteins were extracted, and analyzed by immunoblotting for RIG-I. Average peak densities of RIG-I were normalized to β-actin and are
presented as mean + SEM. Immunoblots are representative of two independent animal experiments. FIGURE 5 | Double-stranded RNA (dsRNA) induces retinoic acid-inducible gene I (RIG-I) expression and phosphorylation of p65 and TANK binding-kinase-1 (TBK1)
in endometrial stromal cells. Statistical Analysis Depletion of TLR3, using siRNA, resulted in reduced RIG-I pro
tein expression in Poly(I:C) transfected stromal cells (Figure 6A). Furthermore, depletion of IRF3, a signaling molecule down
stream of TLR3, resulted in reduced RIG-I protein expression
(Figure 6A). Whereas, depletion of TLR3 or TRAF3 resulted in
reduced IL-6 accumulation in Poly(I:C) transfected stromal cells
(Figures 6B,C). This indicates that TLR3 and IRF3 are important
signaling pathways for RIG-I expression. However, TRAF3 is Poly(I:C) binds to and triggers the activation of the RNA
sensors endosomal TLR3 and RIG-I (also known as DDX58),
among others (1). Bovine endometrial cells are known to express FIGURE 3 | Double-stranded RNA (dsRNA) induces inflammatory mediator production in endometrial cells in a time-dependent manner. Epithelial [(A,B); n = 3
independent animals] or stromal cells [(C,D); n = 4] were transfected with dsRNA Poly(I:C) (1 µg/ml) for 2–72 h (A–D). Concentrations of IL-6 (A,C) or IL-8
(B,D) in supernatants were measured by enzyme-linked immunosorbent assay. Data are presented as mean + SEM, and analyzed by two-way analysis of variance,
using the post hoc Dunnett’s multiple comparison test to compare treatment to control at each time-point, ***P < 0.001, **P < 0.01, *P < 0.05. FIGURE 3 | Double-stranded RNA (dsRNA) induces inflammatory mediator production in endometrial cells in a time-dependent manner. Epithelial [(A,B); n = 3
independent animals] or stromal cells [(C,D); n = 4] were transfected with dsRNA Poly(I:C) (1 µg/ml) for 2–72 h (A–D). Concentrations of IL-6 (A,C) or IL-8
(B,D) in supernatants were measured by enzyme-linked immunosorbent assay. Data are presented as mean + SEM, and analyzed by two-way analysis of variance,
using the post hoc Dunnett’s multiple comparison test to compare treatment to control at each time-point, ***P < 0.001, **P < 0.01, *P < 0.05. August 2017 | Volume 8 | Article 996 Frontiers in Immunology | www.frontiersin.org 6 TLR3 RIG-1 Endometrium Carneiro et al. FIGURE 4 | Double-stranded RNA (dsRNA) induces retinoic acid-inducible gene I (RIG-I) expression in endometrial stromal cells in a time- and
concentration-dependent manner. Epithelial (A) or stromal cells (B) were transfected with dsRNA Poly(I:C) (1 µg/ml) for 2–72 h (A,B) or in increasing concentrations
for 24 h (C). Total cell proteins were extracted, and analyzed by immunoblotting for RIG-I. Average peak densities of RIG-I were normalized to β-actin and are
presented as mean + SEM. Immunoblots are representative of two independent animal experiments. Statistical Analysis Stromal cells (A,B,C) were transfected with Lipid A (0.1 µg/ml), CpG DNA (1 µg/ml), ssRNA (1 µg/ml), dsRNA Poly(I:C) (low-molecular
weight; 1 µg/ml), or Poly(I:C) (high-molecular weight; 1 µg/ml) for 24 h. Total cell proteins were extracted, and analyzed by immunoblotting for RIG-I (A), p65 or
phosphorylated p65 (S536) (B), and phosphorylated TBK1 (S172) (C). Average peak densities of RIG-I, phosphorylated p65, or phosphorylated TBK1 proteins were
normalized to β-actin and are presented as mean + SEM. Immunoblots are representative of two independent animal experiments. FIGURE 5 | Double-stranded RNA (dsRNA) induces retinoic acid-inducible gene I (RIG-I) expression and phosphorylation of p65 and TANK binding-kinase-1 (TBK1)
in endometrial stromal cells. Stromal cells (A,B,C) were transfected with Lipid A (0.1 µg/ml), CpG DNA (1 µg/ml), ssRNA (1 µg/ml), dsRNA Poly(I:C) (low-molecular
weight; 1 µg/ml), or Poly(I:C) (high-molecular weight; 1 µg/ml) for 24 h. Total cell proteins were extracted, and analyzed by immunoblotting for RIG-I (A), p65 or
phosphorylated p65 (S536) (B), and phosphorylated TBK1 (S172) (C). Average peak densities of RIG-I, phosphorylated p65, or phosphorylated TBK1 proteins were
normalized to β-actin and are presented as mean + SEM. Immunoblots are representative of two independent animal experiments. August 2017 | Volume 8 | Article 996 7 Frontiers in Immunology | www.frontiersin.org TLR3 RIG-1 Endometrium Carneiro et al. FIGURE 6 | Inflammatory mediators moderate double-stranded RNA (dsRNA)-dependent retinoic acid-inducible gene I expression, and IL-6 and IL-8 production in
endometrial stromal cells. Stromal cells (A,B,C) were cultured for 24 h in medium plus scrambled short interfering RNA (siRNA) (S) or media containing dsRNA
Poly(I:C) (1 µg/ml). In each independent set of experiments, cells received vehicle plus scrambled siRNA control (S), vehicle plus siRNA targeting Toll-like receptor-3,
interferon regulatory factor 3, TRAF3, or MYD88 18 h before 24 h transfected Poly(I:C) treatment. Concentrations of IL-6 (B) or IL-8 (C) in supernatants were
measured by enzyme-linked immunosorbent assay. Data represent five independent animal experiments. Data are presented as mean + SEM, and analyzed by
two-way analysis of variance, using the post hoc Dunnett’s multiple comparison test to compare siRNA plus Poly(I:C) to S plus Poly(I:C), ***P < 0.001, **P < 0.01,
*P < 0.05. FIGURE 6 | Inflammatory mediators moderate double-stranded RNA (dsRNA)-dependent retinoic acid-inducible gene I expression, and IL-6 and IL-8 production in
endometrial stromal cells. Statistical Analysis Stromal cells (A,B,C) were cultured for 24 h in medium plus scrambled short interfering RNA (siRNA) (S) or media containing dsRNA
Poly(I:C) (1 µg/ml). In each independent set of experiments, cells received vehicle plus scrambled siRNA control (S), vehicle plus siRNA targeting Toll-like receptor-3,
interferon regulatory factor 3, TRAF3, or MYD88 18 h before 24 h transfected Poly(I:C) treatment. Concentrations of IL-6 (B) or IL-8 (C) in supernatants were
measured by enzyme-linked immunosorbent assay. Data represent five independent animal experiments. Data are presented as mean + SEM, and analyzed by
two-way analysis of variance, using the post hoc Dunnett’s multiple comparison test to compare siRNA plus Poly(I:C) to S plus Poly(I:C), ***P < 0.001, **P < 0.01,
*P < 0.05. important for IL-6 production in stromal cells, but is not involved
in the expression of RIG-I. the tested inflammatory cytokines from EVOCs. In epithelial
cells, IL-6 was reduced and IL-8 increased upon treatment with
BoHV-4. BoHV-4 increased stromal IL-8 production, but there
was no observed change in IL-6. These data indicate that endo
metrial cells initiate differing innate immune signaling pathways,
dependent on the specific viral challenge. All viruses, except negative-strand RNA viruses, generate
dsRNA during genome replication, a TLR3 ligand (23). As TLR3
was important for IL-6 production in response to transfected
Poly(I:C), we next investigated the influence of TLR3 on inflam
matory mediator production in response to viable BoHV-4 or
BVDV (Figures 7A,B). Accordingly, depletion of TLR3 using
siRNA resulted in reduced IL-6 production in response to BVDV
(Figure 7A). Finally, to investigate whether BoHV-4 or BVDV
induce increased expression of RIG-I in stromal cells, stromal
cells were exposed to viable virus for 96 h. BoHV-4 induced
an increase in RIG-I expression, whereas RIG-I expression was
reduced in BVDV treated cells (Figure 7C). i
A previous study showed that endometrial cells co-treated
with LPS and BVDV had altered expression of genes associated
with the innate immune response to viruses, including DDX58
(RIG-I) (8). Our data show that RIG-I is barely detectable in
untreated primary epithelial or stromal cells. However, trans
fection of Poly(I:C), a mimetic of viral dsRNA, induced RIG-I
protein expression in a time- and concentration-dependent man
ner in endometrial stromal, but not epithelial cells. Treatment of
stromal cells with viable BoHV-4 also induced increased RIG-I
levels. Although BoHV-4 is a DNA virus, all viruses, except
negative-stranded RNA viruses, have a dsRNA stage in their
lifecycle (23). DISCUSSION Stromal cells (A,B) were cultured for
72 h in medium plus scrambled short interfering RNA (siRNA) (S) or media containing bovine herpesvirus-4 (BoHV-4) or bovine viral diarrhea virus (BVDV) [at 1
multiplicity of infection (m.o.i.)]. In each independent set of experiments, cells received vehicle plus scrambled siRNA control (scramble), vehicle plus siRNA targeting
TLR3 (siTLR3) 18 h before 72 h virus treatment. Concentrations of IL-6 (A) or IL-8 (B) in supernatants were measured by enzyme-linked immunosorbent assay. Data
represent three independent animal experiments. Data are presented as mean + SEM, and analyzed by two-way analysis of variance, using the post hoc Dunnett’s
multiple comparison test to compare siRNA plus virus to S plus virus, ***P < 0.001. Stromal cells (C) were cultured for 96 h in medium containing lipopolysaccharide
(1 µg/ml), BoHV-4, or BVDV (at 1 m.o.i.). Total cell proteins were extracted, and analyzed by immunoblotting for retinoic acid-inducible gene I and to β-actin. Our data does not establish a direct link between BoHV-4,
dsRNA, and RIG-I expression. For example, in the first few
hours of stromal infection with cytomegalovirus, a herpesvirus,
TLR independent IFN-I responses were dependent on cGAS,
STING, and IRF3 signaling (46). Thus, multiple pathways
are involved in controlling early viral replication in stromal
cells in vivo and there is a possibility that other viral PAMPs
or components may be capable of inducing RIG-I expression
through other PRRs. For instance, unmethylated viral CpG-
DNA and viral ssRNA stimulate TLR9- and TLR7-dependent
signaling pathways, respectively (30, 40). However, our data
indicate that transfected dsDNA or ssRNA did not induce
increased RIG-I in endometrial cells. DNA is also detected
intracellularly via AIM2, which initiates the formation of the
inflammasome complex, to orchestrate mature IL-1β release
from cells (20). Our results show a marginal increase in
IL-1β in response to the DNA virus BoHV-4, but a significant
increase in IL-1β from BVDV, an RNA virus. As well as RIG-I,
the RLR family includes the cytoplasmic sensor MDA-5, which
is widely expressed in many cell types (47). In a previous study,
expression of several IFN-inducible genes, including IFIH1,
were significantly increased in cows suffering a severe negative
energy balance status (48). Whether this is a result of increased
viral load in these cows is unclear. DISCUSSION Bovine herpesvirus-4 and BVDV cause uterine disease in cattle,
often resulting in reduced fertility, or abortion of the fetus, respec
tively (11). Countering viral infections requires coordination
of the innate immune system by host cells, including pathways
initiated by PRRs and the appropriate production of cytokines,
chemokines, and IFNs. In this study, we demonstrate that BVDV
induced cytokine and chemokine production in EVOCs and
endometrial epithelial cells, and IL-6 production in stromal
cells. Whereas, BoHV-4 did not induce accumulation of any of Infection with RNA viruses induces cytokine and chemokine
production in a TLR-dependent manner (41, 42). Because RNA
is a universal viral molecular pattern, TLR3 has been assumed to
have a central role in the host response to most viruses (43). For
example, infection with West Nile virus, an ssRNA virus, initiates
an inflammatory response through TLR3, as Tlr3−/− mice are more
resistant to infection with the virus (44). Bovine endometrial cells
increase gene expression of TLR3, complement, and chemotactic August 2017 | Volume 8 | Article 996 Frontiers in Immunology | www.frontiersin.org 8 Carneiro et al. TLR3 RIG-1 Endometrium FIGURE 7 | Toll-like receptor (TLR)-3 moderate’s virus induced inflammatory mediator production in endometrial stromal cells. Stromal cells (A,B) were cultured for
72 h in medium plus scrambled short interfering RNA (siRNA) (S) or media containing bovine herpesvirus-4 (BoHV-4) or bovine viral diarrhea virus (BVDV) [at 1
multiplicity of infection (m.o.i.)]. In each independent set of experiments, cells received vehicle plus scrambled siRNA control (scramble), vehicle plus siRNA targeting
TLR3 (siTLR3) 18 h before 72 h virus treatment. Concentrations of IL-6 (A) or IL-8 (B) in supernatants were measured by enzyme-linked immunosorbent assay. Data
represent three independent animal experiments. Data are presented as mean + SEM, and analyzed by two-way analysis of variance, using the post hoc Dunnett’s
multiple comparison test to compare siRNA plus virus to S plus virus, ***P < 0.001. Stromal cells (C) were cultured for 96 h in medium containing lipopolysaccharide
(1 µg/ml), BoHV-4, or BVDV (at 1 m.o.i.). Total cell proteins were extracted, and analyzed by immunoblotting for retinoic acid-inducible gene I and to β-actin. FIGURE 7 | Toll-like receptor (TLR)-3 moderate’s virus induced inflammatory mediator production in endometrial stromal cells. Frontiers in Immunology | www.frontiersin.org REFERENCES 10. Lanyon SR, Hill FI, Reichel MP, Brownlie J. Bovine viral diarrhoea:
pathogenesis and diagnosis. Vet J (2014) 199(2):201–9. doi:10.1016/j. tvjl.2013.07.024 1. Schlee M, Hartmann G. Discriminating self from non-self in nucleic acid
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mi.2015.131 6. FUNDING This work was funded by the United Kingdom Biotechnology and
Biological Sciences Research Council (BBSRC; Grant number
BB/1017240/1). This work was funded by the United Kingdom Biotechnology and
Biological Sciences Research Council (BBSRC; Grant number
BB/1017240/1). i
In conclusion, we report that bovine endometrial cells are
capable of detecting and responding to virus, and their PAMPs,
through TLR3 and RIG-I. The relative contribution of PRRs
in the innate defense of endometrial cells to viruses requires
further study to delineate the specific roles each contributes. However, from our data it would appear that the response
to viruses and their ligands requires RIG-I in a coordinated
response orchestrated by TLR3, at least in regard to detection
of viral dsRNA. DISCUSSION Unfortunately, in the present
study, we were unable to find a suitable bovine specific MDA-5
antibody to use, and ELISAs specific for bovine type I IFNs are
unavailable. and TRIM factors in response to BVDV (8). However, despite
BVDV inducing increases in inflammatory cytokine production
in EVOCs, epithelial, and stromal cells, in our study increased
RIG-I protein was not evident in BVDV treated stromal cells. This may indicate that endometrial cells use pathways other than
RIG-I, downstream of TLR3, to initiate inflammatory cytokine
production in response to BVDV. p
p
Unlike signaling pathways initiated by all other TLRs, TLR3
does not recruit the adaptor MyD88, but solely depends on the
adaptor TRIF. The TRIF signaling pathway leads to the activation
of NF-κB and IRF3, key transcription factors, with roles in innate
immunity (45). An essential step for IRF3 activation is mediated
by the recruitment of TBK1 to TRIF. Our data demonstrate that
Poly(I:C) induces phosphorylation of NF-κB and TBK1, suggest
ing a functional TLR3 pathway in endometrial stromal cells. This
is further evidenced by using siRNA, as depletion of TLR3 or
IRF3 resulted in reduced RIG-I, indicating an importance for this
pathway in RIG-I upregulation. Furthermore, depletion of TLR3
reduced Poly(I:C) induced IL-6 and IL-8 accumulation, and
BVDV induced IL-6 in stromal cell supernatants, demonstrat
ing the importance of this pathway in cytokine and chemokine
production in response to virus. Interestingly, although IRF3
was important for RIG-I expression, depletion of IRF3 did not
affect IL-6 or IL-8 production. However, we cannot exclude the
contribution of other DNA sensors in endometrial stromal cell
signaling. Thus, multiple pathways are probably involved in
controlling early viral replication in stromal cells in vivo. August 2017 | Volume 8 | Article 996 Frontiers in Immunology | www.frontiersin.org 9 Carneiro et al. Carneiro et al. TLR3 RIG-1 Endometrium ETHICS STATEMENT Replication deficient BoHV-4 viruses are still able to infect
endometrial epithelial and stromal cells, but the virus does
not induce the production of proinflammatory cytokines or
chemokines. This may suggest that the virus is only detected once
viral replication occurs. Only then, once dsRNA is produced, can
the cells initiate an innate immune response via TLR3 and/or
RIG-I. As RIG-I is a cytosolic receptor, stroma may be particularly
vulnerable to BoHV-4 CPE as it takes up to 96 h for increased
RIG-I protein to become evident. Other factors may also play a
part in the pathogenesis of viral infection, other than PRRs. For
example, studies show that the BoHV-4 IE2 (ORF50/Rta) gene
transactivates the CXCL8 (IL8) gene promoter, either directly or
indirectly during BoHV-4 infection (49). Therefore, IL-8 produc
tion may actually be of benefit to the virus. Uteri with no gross evidence of genital disease or microbial infec
tion were collected from cattle processed as part of the normal
work of an abattoir. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online at http://journal.frontiersin.org/article/10.3389/fimmu. 2017.00996/full#supplementary-material. AUTHOR CONTRIBUTIONS JC, IMS, VL, and GD designed the study. JC and IMS wrote the
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and Cronin. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in
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73(4):372–82. doi:10.1111/aji.12344 August 2017 | Volume 8 | Article 996 Frontiers in Immunology | www.frontiersin.org 11
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Detection and Isolation of DoS and Integrity Cyber Attacks in Cyber-Physical Systems with a Neural Network-Based Architecture
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Detection and Isolation of DoS and Integrity Cyber
Attacks in Cyber-Physical Systems with a Neural
Network-Based Architecture es 1
, Diego Martínez-Castro 1, Vrani Ibarra-Junquera 2 and Apolinar González-Potes 2,3,* Carlos M. Paredes 1
, Diego Martínez-Castro 1, Vrani Ibarra-Junquera 2 and Apolinar Gonzál Carlos M. Paredes 1 1
Departamento de Automática y Electrónica, Universidad Autónoma de Occidente, Cali 760030, Colombia;
cmparedes@uao.edu.co (C.M.P.); dmartinez@uao.edu.co (D.M.-C.) 1
Departamento de Automática y Electrónica, Universidad Autónoma de Occidente, Cali 760030, Colombia;
cmparedes@uao.edu.co (C.M.P.); dmartinez@uao.edu.co (D.M.-C.)
2
Laboratorio de Agrobiotecnología, Universidad de Colima, Colima 28400, Mexico; vij@ucol.mx
3
Facultad de Ingeniería Mecánica y Eléctrica, Universidad de Colima, Colima 28400, Mexico
*
Correspondence: apogon@ucol.mx 1
Departamento de Automática y Electrónica, Universidad Autónoma de Occidente, Cali 760030, Colombia
cmparedes@uao.edu.co (C.M.P.); dmartinez@uao.edu.co (D.M.-C.)
2
Laboratorio de Agrobiotecnología, Universidad de Colima, Colima 28400, Mexico; vij@ucol.mx
3
Facultad de Ingeniería Mecánica y Eléctrica, Universidad de Colima, Colima 28400, Mexico
*
Correspondence: apogon@ucol.mx Abstract: New applications of industrial automation request great flexibility in the systems, sup-
ported by the increase in the interconnection between its components, allowing access to all the
information of the system and its reconfiguration based on the changes that occur during its opera-
tions, with the purpose of reaching optimum points of operation. These aspects promote the Smart
Factory paradigm, integrating physical and digital systems to create smarts products and processes
capable of transforming conventional value chains, forming the Cyber-Physical Systems (CPSs). This
flexibility opens a large gap that affects the security of control systems since the new communication
links can be used by people to generate attacks that produce risk in these applications. This is a
recent problem in the control systems, which originally were centralized and later were implemented
as interconnected systems through isolated networks. To protect these systems, strategies that have
presented acceptable results in other environments, such as office environments, have been chosen. However, the characteristics of these applications are not the same, and the results achieved are not as
expected. This problem has motivated several efforts in order to contribute from different approaches
to increase the security of control systems. Based on the above, this work proposes an architecture
based on artificial neural networks for detection and isolation of cyber attacks Denial of Service (DoS)
and integrity in CPS. Simulation results of two test benches, the Secure Water Treatment (SWaT)
dataset, and a tanks system, show the effectiveness of the proposal. electronics
Citation: Paredes, C.M.; Martínez-
Castro, D.; Ibarra-Junquera, V.;
González-Potes, A. Detection and
Isolation of DoS and Integrity Cyber
Attacks in Cyber-Physical Systems
with a Neural Network-Based
Architecture. Electronics 2021, 10, 2238. https://doi.org10.3390/electronics
10182238 Academic Editor: Arman Sargolzaei Received: 30 July 2021
Accepted: 31 August 2021
Published: 12 September 2021 Keywords: anomaly detection; anomaly isolation; artificial neural networks; Cyber Physical System Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Detection and Isolation of DoS and Integrity Cyber
Attacks in Cyber-Physical Systems with a Neural
Network-Based Architecture Regarding the SWaT dataset, the
scores obtained from the recall and F1 score metrics was 0.95 and was higher than other reported
works, while, in terms of precision and accuracy, it obtained a score of 0.95 which is close to other
proposed methods. With respect to the interconnected tank system, scores of 0.96, 0.83, 0.81, and 0.83
were obtained for the accuracy, precision, F1 score, and recall metrics, respectively. The high true
negatives rate in both cases is noteworthy. In general terms, the proposal has a high effectiveness in
detecting and locating the proposed attacks. Citation: Paredes, C.M.; Martínez-
Castro, D.; Ibarra-Junquera, V.;
González-Potes, A. Detection and
Isolation of DoS and Integrity Cyber
Attacks in Cyber-Physical Systems
with a Neural Network-Based
Architecture. Electronics 2021, 10, 2238.
https://doi.org10.3390/electronics
10182238 1. Introduction Cyber Physical Systems (CPSs) emerge from the attempts to unify the emerging
applications of embedded computers and communication technologies used to monitor,
control, as well as generate actions on physical elements to fulfill with a specific task [1],
and they have an important impact on different sectors [2]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). The different parts of the system are usually interconnected using communication
networks to share information and data that interact with each other and, sometimes, cloud
computing services [3–5]. CPSs can be represented in layers, as shown in Figure 1. The
first is the physical layer, where the physical infrastructure of the system, sensors, and
actuators are located, with the objective of monitoring and controlling physical processes. https://www.mdpi.com/journal/electronics Electronics 2021, 10, 2238. https://doi.org/10.3390/electronics10182238 Electronics 2021, 10, 2238 2 of 28 2 of 28 The second is the network layer, which implements the transmission data and allows the
interaction between the physical layer and the cybernetic layer. Finally, a cybernetic layer
allows the abstractions of the received data, as well as the interaction between networks,
devices, and the physical infrastructure [6]. Figure 1. Architecture of a CPS. Figure 1. Architecture of a CPS. Society currently relies on multiple automatic systems supported by CPSs. These
applications are focused in contexts, such as industrial, health, and environmental, among
others [7,8]. Security and reliability are fundamental requirements in these systems. Cyber
attacks can generate inappropriate behaviors and catastrophic impacts on the physical
world, causing damage to both the system infrastructure and industrial products and
even threaten human lives [9]. Examples, such as attacks on smart grids, aviation systems,
water plants, chemical plants, and oil and natural gas distribution systems, are becom-
ing increasingly high [10–14]. The above has caused this research area to be active in
recent years. y
Therefore, there must be mechanisms to detect the occurrence anomalies to avoid
exploiting vulnerabilities in the devices connected to the system network. Real-time
detection is very important in order to ensure reliability and security in these systems,
where sensors are prone to malicious attacks. For this reason, detection systems are often
used, such as Intrusion Detection Systems (IDS), which monitor data traffic to identify and
protect systems from these eventualities. 1. Introduction Based on detailed analysis of network traffic and
device usage, IDSs seek to evaluate this information to identify unwanted events. IDSs do
this by carrying out three stages: monitoring, analysis, and detection. Monitoring relies
on a sensor network or host-based sensors, the analysis stage is based on any method to
identify and extract features, and the detection stage relies on anomaly detection [15,16]. Within these can be highlighted: [17] the methods based on traditional information
technologies, where network traffic analysis is used to detect anomalies [18–26]; and model-
based methods, where detection is performed by comparing the system actual output with
an expected value [4,27–31]. According to reference [16,32], host-based IDS methods operate on the data collected
from the individual parts of the computer systems and can detect internal changes and
determine which processes and/or users are involved in malicious activities, which can be
not significant with some devices; thus, this method sometimes fails. Whereas a network-
based IDS will detect malicious packets as they enter your network or unusual behavior on
your network, such as flooding attacks, more traditional IDS can do it on one channel or
across the network. These monitor the entire network traffic to detect known or unknown
attacks using techniques based on anomalies, signatures, and specifications [16,33,34]. Hence, IDSs help to avoid critical consequences and assist in making appropriate decisions
when system events occur by performing two main tasks: attack detection, which decides
whether or not an anomaly has occurred; and attack isolation, which decides which
elements of the system are being affected by the unwanted. In such a way, the purpose of this research is to present the design of an architecture
that allows detecting and isolating attacks that may occur between the elements of the
physical layer and the controller, generating alerts that allow detection and localization
of the origin of the cyber attacks. For this, a new architecture was proposed for the Electronics 2021, 10, 2238 3 of 28 3 of 28 detection and isolation of attacks using techniques based on artificial intelligence. The
proposal integrates two approaches: regression and classification. The first approach
allows generating models that describe the behavior of the real process to estimate the
outputs by using process input data, obtaining in this way the model to be compared
with the real process values in order to detect and isolate the attack. 1. Introduction The second approach
allows generating detection systems to carry out the detection and isolation of attacks. The
proposal was subjected to two test benches, obtaining better results than those reported in
previous works. The contributions of this paper are as follows: •
The design of an architecture using one-dimensional convolutional neural networks to
detect and isolate cyber-attacks that involve the elements of the physical layer and the
controller of a CPS, generating alerts to detect and locate the origin of the cyber attack. •
The architecture proposed is an architecture based on the process information, where
the dynamic properties of the process are covered, in order to evaluate the possibility
of a cyber attack occurring in different parts of the system, without the need to define
a threshold that allows separating normal situations with events where a cyber attack
is possibly occurring. p
y
g
•
The design of the architecture allows detecting and locating the occurrence of cyber
attacks occurring simultaneously in different parts of the system, even when the
attacks are of different types. The remaining sections are structure as follows. Section 2 presents related works. Section 3 describes the problem statement. In Section 4, the attack detection and isolation
method is proposed. Section 5 exposes the results obtained using the method proposed in
two test benches. Finally, in the last section, we present the conclusions. 2. Related Works Therefore, this method generally has high
false-positive rates. On the other hand, the methods based on specifications use a set of
rules and thresholds that define the expected behavior of the different components of the
network. It has the same purpose as anomaly-based methods, with the difference that this
method is specified manually by an expert who determines the specifications. Manually
defined specifications typically provide low false-positive rates versus anomaly-based
detection and do not require training steps because it can be used immediately. However,
these methods cannot be adapted to different environments and can be time-consuming to
adjust and error-prone [33]. Other authors have developed state observers for detection, such as the Luenberger
Observer (LO), while the isolation process is realized by structured residues generated
using Unknown Input Observers (UIOs) [37–40]. These methods present drawbacks
because the detection of anomalies is realized by a comparison of a fixed threshold defined
by a historical data of normal behavior, with the difference between the variables of the
actual process and the values generated by an estimated model. Then, it can lead to a
considerable rate of false positives and false negatives. The above is because, for the design
of the observer banks, the knowledge of the parameters and the dynamics of the system is
used, which sometimes can be significantly different of the real system performance. So,
both proposals are limited by the knowledge of the process, such as the definition of the
threshold, which, in real situations, it may not be easy to model accurately. In the last few years, data-driven methods have been employed to detect cyber
attacks [18–23,25,41]. These methods have presented good performance to find models
of processes that even present quite pronounced non-linear dynamics. Machine learning
technology is one of the data-driven methods emerging as a method to detect attacks in
these systems [23,26,42–50]. Random Forest-based algorithms have been employed recently to detect malicious
behavior by using databases; in this case, binary classification is applied to classify whether
the content of a packet is malicious or not. This method reduces computational cost but
does not guarantee high accuracy [51]. In this way, it is not possible to identify which
task transmitted the packet, and it does not allow specifying the type of attack [15,16]. From another point of view, in Reference [52], a scheme was proposed to protect remote
patient monitoring systems against DoS attacks. 2. Related Works Protection systems in industrial processes have used strategies that have presented
good performance in other environments, such as office environments. However, the
characteristics of these applications are not the same, so the results obtained are not as
expected. This is because the availability of equipment in industrial systems is very high;
so, in many cases, a simple solution in corporate environments, such as patching, simply
does not work because the machine is not available to shut down until a planned outage. It is also difficult to predict how a newly introduced patch will affect the operation of a
control system, especially if the patch is not rigorously tested, increasing the organization’s
reluctance to act on potential threats. The implementation of security patches can affect
application performance and, therefore, the stability, availability, and real-time behavior
of machines. Something equivalent occurs with the impact on data traffic through the
communications network associated with solutions that evaluate network traffic, which can
affect delays in control strategies and, in turn, the performance of control loops [35]. This
problem has motivated different projects with the purpose of contributing from different
approaches to increase the security of control systems. In this section, the related works
are described. Some ongoing projects to improve security in these systems have included methods to
provide aspects, such as data confidentiality and authentication, access control, within the
network, and privacy and reliability of applications, as well as the inclusion of security and
privacy policies [36]. Even so, CPSs are vulnerable to multiple attacks aimed at disrupting
the network and modifying process variables, altering its operation. For this reason, new
defense mechanisms designed to detect cyber attacks have been generated. One of the
best known mechanisms is IDS. IDS approaches may be classified as signature-based,
anomaly-based, or specification-based [33]. The signature-based method only detects records that are inside of a database, and it
is highly accurate and effective against known threats, consumes more power, and does not
detect new events [33]. The anomaly-based method is efficient in detecting new attacks [16]
since it compares the system activities in a moment against an usual behavior profile and
generates alerts whenever a threshold defined by the system’s normal behavior is cross [34]. Electronics 2021, 10, 2238 4 of 28 However, anything that does not match normal behavior is considered an intrusion, and
learning all normal behavior is not an easy task. 2. Related Works An attack detection model was established
by developing mechanical learning using decision trees. The model could help to locate
various types of attacks, focusing mainly on flooding attacks, and could be appropriate
to devices with limited memory and processing resources, such as sensors and healthcare
devices. As future work, they propose the possibility of identifying other types of attacks
and even developing a mechanism to block a wide range of attacks. p
g
g
Other approaches have used different artificial intelligence techniques, such as Support
Vector Machines (SVMs), genetic algorithms [32], self-organized networks of ant colonies,
and extreme learning machines, which provide models with very high accuracies applied in
the context of security in computer networks, and especially in the detection of intrusions. The purpose of these techniques is to achieve better intrusion recognition rates, but it is
still noticeable that the false positive rate remains the problem to be approached in all
these studies. Although some technique can reduce the false positive rate, it increases the
training time and classification, which is a relevant index for real-time detection [53]. In Reference [18], an SVM-based algorithm was used to classify normal and abnormal
behavior of data traffic that may be subjected to DoS attacks. This algorithm reaches
good attacks predictions rate with less training time. In Reference [19], a method based
on Principal Component Analysis (PCA) and SVM to detect DoS attacks was presented. The paper analyzes the effects of DoS attacks in a network using TCP protocol. The PCA
algorithm is used in order to filter the interference of the environment to extract the main
features effectively and reduce the dimensioning of information without losing information
from the original data. The results show that the algorithm has high accuracy and a low Electronics 2021, 10, 2238 5 of 28 false positive and false negative rate (FPR and FNR). In the same context, an SVM using a
radial basis kernel function is proposed in Reference [20] to detect attacks in networked
automotive systems. This proposal aims to avoid drawbacks associated with cases in which
there is not an events dataset, or it is probably not sufficiently representative because many
of the possible situations of a system are unknown. However, these techniques are not
suitable for detecting mutations from various attacks. 2. Related Works Advanced techniques, such as Deep Belief Networks (DBN) and Deep Convolutional
Neural Networks (Deep CNN) [54,55], are trained to extract low-dimensional features and
are used to discriminate usual and hacking packets. In Reference [56], an anomaly detector
based on a neural network recurrent Long short-term memory (LSTM) was proposed to
detect attacks with low false alarm rates. These methods have had the best response in
these environments, although the computational costs sometimes are high [20,55]. Thus,
applying machine learning and other artificial intelligent techniques is a challenge be-
cause it requires more memory and computational cost that can affect the performance of
the system. In addition, to validate the proposal, two test benches were used. For the selection
of these datasets, a search was performed that included keywords, such as security in
industrial control systems, detection of faults, anomalies and cyber attacks in control
systems, and design of secure CPSs. From this search, we considered the publications that
had a publication time of less than 5 years, as well as the number of times that the datasets
had been used to evaluate the security on CPSs. We also considered the type of attacks that
were implemented, since our approach was to address different types of attacks, including
those with the highest frequency and impact on the control systems found in the CPSs
(integrity and DoS attacks). The first one corresponds to the SWaT dataset, which provides real data from a
simplified version of a real world water treatment plant. This dataset allows researchers
to design and evaluate defense mechanisms for CPSs and contains both network traffic
and data concerning the physical properties of the system [57]. On the other hand, there
is another test bed which consists of three interconnected tanks [58] that has allowed the
validation of different types of detection methods for cyber attacks on CPS. These two test
benches have made it possible to validate different proposals focused on techniques that
allow us, in one way or another, to analyze the detection of cyber attacks [37,42,59–69] and
have made it possible to direct this research to improve the proposed proposals. Based on this review, Table 1 summarizes each of the related reports to a set of
characteristics in order to highlight the issues that need to be addressed to improve the
strategies and proposals in the future. Electronics 2021, 10, 2238 6 of 28 Table 1. 2. Related Works Summary of related works. Reference
Main Domain
Technique
Type of Anomaly
Advantages
Limitations
Evaluation
[18]
Mobile networks
SVM, signature and anomaly based methods
DoS attacks
High accuracy to detect normal and anomalous behavior
Only detects DoS attacks
Dataset KDD
[19]
Mobile networks
PCA-SVM
Low rate DoS attacks
High detection rate and low FPR and FNR
Only detects DoS attacks
Simulation
[20]
In-vehicle networks
One-class SVM
Possible errors in the recordings
The proposed methodology could be applied to several fields
TNR below 77% and precision below 76%. Dataset from a real vehicle
[23]
Mobile networks
MLP for intrusion detection
DoS attacks
High accuracy to detect normal and anomalous behavior
Only detects DoS attacks
Dataset KDD
[25]
Heavy duty vehicle system
Gaussian radial basis function neural network
Deception attacks
Can be applied to a variety of nonlinear CPSs
Attacks occur in only one part of the system
Simulation
[26]
Solar Farms
Multilayer LSTM network
Integrity attacks
Accuracy, recall, precision and F1 score are above 90%
Attacks occur in only one part of the system
Simulation
[37]
Three-tank system
Luenberger Observers (LOs) and Unknown
Input Observers (UIOs)
Integrity attacks
Possibility to mitigate the effect of the attack
Attacks occur in only one part of the system. Dependence on
threshold selection. Simulation
[38]
Smart grids
Unknown Input Observers (UIOs)
Integrity attacks
Possibility to mitigate the effect of the attack. Attacks occur in only one part of the system Dependence on
threshold selection. Simulation
[39]
Power systems
Unknown Input Observers (UIOs)
Integrity attacks
Possibility to mitigate the effect of the attack
Attacks occur in only one part of the system. Dependence on
threshold selection
Simulation
[40]
Power systems
Luenberger Observers (LOs) and Unknown
Input Observers (UIOs)
Integrity and DoS attacks
Platform for simulating different types of cyber attacks
Detection depends on the selection of the threshold. Emulation and simulation
[41]
Automotive Brake Systems
Recurrente neural networks
Integrity attacks
High accuracy
The attacks are applied on the same part. Experimental
[42]
Industrial Control Systems
1D CNN and GRU
Integrity attacks
High precision and F1 score. False alarm rate needs to decrease. SWaT Dataset
[50]
Automated Vehicles
LSTM and CNN
Various
Detecting different single anomaly types. In some cases the TPR is low. Experimental. [55]
Heavy-duty gas turbines of combined
cycle power plants
Stacked denoising autoencoder
Various
Real time detection, high TPR and low FPR. Only detects, does not locate. Simulation and data from
real plants. 2. Related Works [56]
Automobile Control Network Data
LSTM
Integrity attacks
High TPR and low FPR. It is required to achieve a practical level to reliably
detect anomalies. Simulation. [59]
Industrial Control System
Genetic algorithms and neural network
Various
High accuracy to locate the sensor under attack. Metrics, such as F1 score and recall, must be improved. SWaT Dataset. [60]
Industrial Control System
Deep Neural Networks
Various
Successfully detects the vast majority of attacks with a low level of
false positives. Metrics, such as F1 score and recall, must be improved. SWaT Dataset. [61]
Industrial Control System
Graphical model-based
Various
High precision. Metrics, such as F1 score and recall, must be improved. SWaT Dataset. [62]
Industrial Control System
SVM and Deep Neural Networks
Various
High precision. Metrics, such as F1 score and recall, must be improved. SWaT Dataset. [63]
Industrial Control System
LSTM and CNN
Various
High precision. Metrics, such as F1 score and recall, must be improved. SWaT Dataset. [64]
Industrial Control System
Lightweight Neural Networks and PCA
Various
Good precision. Metrics, such as F1 score and recall, must be improved. SWaT, BATADAL, and
WADI Dataset. [65]
Networked Control (Three-tank system)
Resilient Tracking Control
Deception and DoS attacks
A combination of attacks can be taken into account to form a
sophisticated and stealthy attack model. High dependence on knowledge of system parameters. Simulation and experimental. [66]
Three-tank system
Model-based fault/attack tolerant
Integrity attacks
Determines when the control input is to be updated again, depending on
the occurrence of the anomaly. High dependence on knowledge of system parameters. Simulation and experimental. Table 1. Summary of related works. Electronics 2021, 10, 2238 7 of 28 Based on the review of the related works, it became evident that there are still chal-
lenges concerning the detection of cyber attacks within the control systems found in the
CPSs. On the one hand, methods must be sought to decrease both the false positive and
false negative rates, and to increase the true positive and true negative rates. This will
improve the overall performance of these detection systems. It is also evident that the
phenomenon of simultaneous attacks has not been addressed in the design of cyber attack
detection systems, which is worrying because these situations can occur very often in the
real world. 2. Related Works Is important to clarify that, within a CPS, there are many points where a cyber-
attack can occur and that can cause different consequences in the system. The emphasis of
this work seeks to design an architecture that allows detecting and locating attacks that
occur between the elements of the physical layer and the controller of a CPS, precisely
in attacks that modify or interrupt the sending of data from one element to another. In
this way, this paper presents the design of an architecture that explores the potential of
convolutional neural networks to extract features and, thus, to determine whether there
is an event related to the possibility of a cyber attack occurring. This approach may have
a closer approach to the implementation in real cases in which there is a high degree of
uncertainty in the process models, since, on many occasions, the way to detect an anomaly
or not is done under a process of comparison between estimated values and the real values
of the process, which is subsequently evaluated by a threshold. In our proposal, this
evaluation is carried out in an intrinsic way by the architecture based on convolutional
neural networks, generating a better performance than current works, as well as shows
promising results in the detection and isolation of simultaneous attacks. 3. Problem Statement Several control applications supported in these systems can be labeled as safety critical
in relation to the fulfillment of strict real time deadlines, associated with the generation of
actions from the interaction between the computational systems and the physical systems
related to the application, because the non-fulfillment of these requirements can cause
irreparable damage to the physical system being controlled, as well as to the people
depending on it [70]. Additionally, measurements and control actions can be altered
while being transmitted through communication networks, thus requiring new control
algorithms or design architectures, which, in the presence of adverse situations, can bring
the system to safe and stable states [71,72]. The proposal presented in this work focuses in
the detection and isolation of DoS and integrity cyber attacks on CPSs, specifically on the
exchange of information between sensors, actuators, and controllers. The approach realized
is based in the fault detection and isolation systems for what anomalies are represented as a
variation of the system parameters [58]. Then, any control system where its control signals
and/or measured variables are susceptible to be attacked can be modeled as a combination
of the two models defined in (1) and (2). x(k + 1) = Ax(k) + Bu(k) + Fa fa(k),
(1)
y(k) = Cx(k) + Fs fs(k),
(2) x(k + 1) = Ax(k) + Bu(k) + Fa fa(k),
(1) (1)
(2) (1) y(k) = Cx(k) + Fs fs(k),
(2) (2) where x(k) represents the state vector, x(k) ∈Rn×1, y(k) is the output vector, y(k) ∈Rp×1,
u(k) is the control action, u(k) ∈Rm×1, matrix A is the state matrix, A ∈Rn×n, B is the
input matrix, B ∈Rn×m, C is the output matrix, C ∈Rp×n, D is the feedthrough matrix,
D ∈Rp×m, Fa = B, and fa = (Γ −I)U + Uf0. ΓU and Uf0, represent the effect of a
multiplicative anomaly and an additive effect in the control action, respectively. DoS and
integrity attacks are visible as anomalies on the control action. If the i-th control action
is attacked, then the matrix Fa corresponds to the i-th column of the matrix B, and fa
corresponds to the magnitude of the attack that directly affects the controller. 3. Problem Statement Electronics 2021, 10, 2238 8 of 28 Similarly, if the i-th sensor is attacked, the matrix Fs is the i-th row of the matrix C, and
the vector of attacks is fs, which represents the magnitude of the effect produced in the
i-th sensor. The problem with traditional methods based on mathematical models that describe
the behavior of the system is that these models are dispensable of the complete knowledge
of the system parameters, and the adaptation in real conditions can cause the overall
performance to decrease. Because of this, we intend to address this problem from models
based on artificial neural networks, precisely in one-dimensional convolutional neural
networks, which have shown very promising results in fields where patterns are sought to
identify a class. Modeling of the Cyber Attack Measurements of process signals and control action values are critical to the proper
functioning of a control system, and its modification by cyber attacks can produce instability
in the control system [73,74]. A cyber attack by data manipulation is called an integrity
attack, modeled by (3), and an attack that results in a prolonged loss of these signals is
called a type DoS attack, which is modeled by (4). yi(k) = yi(k) + yi(k)a,
(3)
yi(k) = yi(k)ts−1,
(4) (3) (4) where yi(k) corresponds to the sensor measurement that reaches the controller in the k-time,
yi(k) corresponds to the sensor measurement before being transmitted to the controller in
the k-time, and yi(k)a is a vector injected by the attackers which changes the yi(k) measure
in the k-time. yi(k)ts−1 corresponds to the measurement before the start of the DoS attack. The time interval for the occurrence of the attack is defined by τa = [ts te]. For the development of the proposal, it is assumed that any sensor can be affected
by any type of attack, integrity, or DoS. Additionally, the attacks may occur at any time in
various parts of the system. The last premise is significant to note because simultaneous
attacks are less discussed in previous works; thus, depending on the type of attack carried
out on the system, output (2) may take the form of (3) and/or (4). Architecture Proposed for the Detection and Isolation of Cyber Attacks in CPS Architecture Proposed for the Detection and Isolation of Cyber Attacks in CPS The architecture proposed is presented in Figure 2. This architecture includes a pre-
diction model which uses an input dataset x0, x1, . . . , xk−1 to estimate outputs ˆy1, ˆy2, . . . , ˆyk
(these datasets will depend specifically on the type of data available from the process), and
these values are used to obtain the residual signal res(k), as is shown in (5). These signals
are used by a classifier to detect anomalies presents in the process. Input
data
Predicting
model
Output
process
Classification
model
Output
data
xk−1
ˆyk
res(k)
yk
Figure 2. General architecture model to detect and isolate cyber attack. Output
data Figure 2. General architecture model to detect and isolate cyber attack. As the characteristics of the signals in a specific process are different then values with
different magnitude could affect the classifier training procedure, therefore, all input data
to the classifier are normalized using its mean and standard deviation to obtain a z-score
for each one as is shown in (7). (7) z = |x −µ|/σ,
(7) where x are the input data, µ is the mean, and σ is the standard deviation. Although the architecture presented is general, it is a base for selecting different
types of machine learning for the prediction and classification stages. The idea is to use
deep neural networks to extract patterns that allow the detection of cyber attacks (such
as LSTM or CNN 1-dimensional). As was not included a method to find spatial-temporal
correlations to detect cyber attacks, it is expected that neural networks will be able to carry
out this task implicitly. p
y
The architecture can be detailed as follows for a specific CPS, shown in Figure 3. A
model of the dynamics of the process generates the outputs signals x(k)s which correspond
to the reconstruction of all the states (it is assumed that the outputs are the process states
or some linear combination of them, although it can be extended to non-linear cases). 4. Attack Detection and Isolation Method In the context of this work, most cyber attack detection methods use the available
data to develop a model that determines the usual behavior of the system. Then, by a
comparison between the estimated outputs of the model and the actual process outputs,
determination of if the behavior of the system is normal or if a cyber attack is taking place. To isolate the attack, which is nothing more than locating the part of the system that is
being affected directly by the cyber attack, decoupled models of the system are developed
that are susceptible only to cyber attacks that occur in specific parts of the system. The procedure to perform this task can be grouped into three steps. Firstly, the
generation of a residual signal is realized, and this process consists of comparing the
measured output with an estimated output. This signal is denoted as residual signal, res(k),
this is described in (5). (5) res(k) = y(k) −ˆy(k),
(5) where y(k) are the set of output measures of the actual process, and ˆy(k) are the set of
outputs estimated. The second step corresponds to the evaluation of the residual; in this
case, a comparison of the residuals is made with a predefined threshold, as is shown in (6). |res(k)| > τthresholds. (6) (6) The thresholds are obtained from data in which the attacks have been presented, thus
allowing their detection and isolation. Finally, a decision-making process is carried out
through indicators. Electronics 2021, 10, 2238 9 of 28 9 of 28 These steps involve the use of residuals that should take values close to 0 in situations
where the system is not being attacked. On the other hand, when an attack is present, the
residual signals must have values other than 0. Although a single residual signal can alert or detect a cyber attack, a set of residuals is
required to isolate it. Then, to locate the origin of the cyber attack, it is necessary that some
residues be sensitive only for a particular part of the system. The above implies that the set
of residuals must be independent of other cyber attacks defined. In this way, to isolate a
cyber attack, a structured set of residuals is considered, where each residual vector can be
used to detect a cyber attack in a specific place of the system. 4. Attack Detection and Isolation Method In the architecture model proposed in this work, it is emphasized that second step
will be an implicit step because the architecture based on artificial neural networks will
interpret the input data generating intrinsic characteristics that will allow the evaluation to
detect and isolate the attacks. 5. Case Studies and Results Analysis Two test benches were used to evaluate the performance of the proposed architecture,
the SWaT dataset [77,78] and an interconnected tank [58]. Architecture Proposed for the Detection and Isolation of Cyber Attacks in CPS In
order to isolate the attack, there is a set of neural network models that relate the process
states with their respective control actions for generate states that are decoupled from each
other (x(k)d1,2,...x); in this way, it is possible to isolate the attack in a way equivalent to the
use of UIOs, but with the advantage that neural networks allow addressing the uncertainty
in the representations. With this set of neural networks, res(k) is generated. p
g
Detection and isolation functions are implemented using artificial neural networks,
which use the process states x(k), the control actions u(k), the reference signals r(k), the
residual signals res(k), and the signals generated by the predicting model. 10 of 28 Electronics 2021, 10, 2238 Process
Controller
Neural network
estimated states
Neural network
decoupled states
Neural network
decoupled states
Residuals
Neural network
detection and
isolation system
x(k)
x(k)2,3,...,n
x(k)1,2,...,n−1
... x(k)
y(k)
u(k)
u(k)
r(k)
x(k)s
x(k)d1
x(k)dn
res(k)
Detection and
isolation
Figure 3. Architecture based on neural networks for the detection and isolation of the cyber attack. Detection and
isolation Neural network
estimated states Figure 3. Architecture based on neural networks for the detection and isolation of the cyber attack. Figure 3. Architecture based on neural networks for the detection and isolation of the cyber attack. Mean squared error (MSE) [75] is adopted as the model’s loss function to train the
predicting model. MSE = 1
n
n
∑
i=1
(xi −ˆxi)2,
(8) (8) where n is the amount of data, xi is the real state, and ˆxi is the estimated state. For the
classifier, the cost function categorical crossentropy (CCE) is used [76] because it is a
single-label multi-class classification problem. where n is the amount of data, xi is the real state, and ˆxi is the estimated state. For the
classifier, the cost function categorical crossentropy (CCE) is used [76] because it is a
single-label multi-class classification problem. JCCE = −
l
∑
q=1
p
∑
k=1
dqk log(yqk). (9) (9) With p classes, training data size of l, the input of xq, where q = 1, 2, . . . , l and yqk
(0 ≤yqk ≤1),k = 1, 2, . . . , p is the estimated probability that belongs to class k, and dqk (0
or 1) becomes the given label (9). 5.1. Secure Water Treatment Dataset-SWaT This dataset was completed by the Singapore University of Technology and Design to
provide researchers with data collected from a complex and realistic ICS environment. The
testbed is a fully operational scale water treatment plant that produces purified water. SWaT
is composed of six main processes corresponding to the physical and control components
of the water treatment plant; each stage (from P1 to P6) is equipped with a certain number
of sensors and actuators. The sensors include flow meters, water level meters, conductivity,
and pH analyzers, among others, while the actuators consist of pumps that transfer water
from one stage to another, pumps that dose chemicals, and valves that control inlet flow. The process is not circular, and P6 water is removed. Sensors and actuators in each stage 11 of 28 Electronics 2021, 10, 2238 11 of 28 are connected to the corresponding PLC (programmable logic controller). This process is
shown in Figure 4. g
g
g
shown in Figure 4. Raw Water
Tank
Pump
P1
LIT101
P101
HCL
NaOCl
NaCl
Static Mixer
FIT201, AIT201
P201
P203
P205
Chemical tanks and dosing pumps
P2
UF Feed Tank
UF Feed Pump
Ultrafiltration
Unit (UF)
P301
LIT301
AIT202
AIT203
DPIT301
P3
RO Feed Tank
RO Feed Pump
Ultraviolet
Dechlorinator
NaHSO3
LIT401
FIT401
P401
AIT402
P4
Cartridge
Filter
RO Boost
Pump
Reverse Osmosis(RO)
Unit
AIT503
P501
P5
AIT504
UF backwash
Tank
Raw Permeate
Tank
UF backwash
Pump
Permeate
Reject
Water recycled
P602
P6
Figure 4. SWaT testbed processes overview [57]. Raw Water
Tank
Pump
P1
LIT101
P101
HCL
NaOCl
NaCl
Static Mixer
FIT201, AIT201
P201
P203
P205
Chemical tanks and dosing pumps
P2
UF Feed Tank
UF Feed Pump
Ultrafiltration
Unit (UF)
P301
LIT301
AIT202
AIT203
DPIT301
P3
RO Feed Tank
RO Feed Pump
Ultraviolet
Dechlorinator
NaHSO3
LIT401
FIT401
P401
AIT402
P4
Cartridge
Filter
RO Boost
Pump
Reverse Osmosis(RO)
Unit
AIT503
P501
P5
AIT504
UF backwash
Tank
Raw Permeate
Tank
UF backwash
Pump
Permeate
Reject
Water recycled
P602
P6
Figure 4. SWaT testbed processes overview [57]. Chemical tanks and dosing pumps
P Chemical tanks and dosing pumps
P Ultraviolet
Dechlorinator Figure 4. SWaT testbed processes overview [57]. Stage P1 controls the flow of raw water by opening or closing a motorized valve that is
connected to the inlet of tank T101. 5.1. Secure Water Treatment Dataset-SWaT By means of the P101 pump, water starts flowing from
T101 through the chemical dosing station in stage P2 and is followed by the ultrafiltration
(UF) process located in stage P3, which eliminates unwanted materials. Similarly, the feed
pump in stage P3 is responsible for supplying the water to the ultrafiltration unit. In the
P5 stage, inorganic impurities are separated by a reverse osmosis process. The output of
the reverse osmosis process is stocked in the permeate tank of stage P6 for its distribution. The P6 stage is also controlling the cleaning of the ultrafiltration membranes in P3 by the
backwashing process. Every certain period of time, the backwash process is triggered by
turning on the backwash pump and is accomplished in under one minute. The backwash
process can alternatively be started by a PLC when the differential pressure sensor value
increases above 0.4, which means that the UF membranes are choked [57,78]. 5.1.2. Data Preparation and Model Training The data from the first dataset is used to generate a model corresponding to the
“Predicting model” block shown in Figure 2. The architecture proposed in this case is based
on a 1D CNN model, as shown in Figure 5. Figure 5. Prediction model for SWaT dataset. Figure 5. Prediction model for SWaT dataset. The input data is composed of 43 characteristics compounded mainly by sensors
measurements, states of the pumps, and valves positions. The first convolution layer
consists of 2 filters, and the kernel size is 3. The 1D average pooling layer has a stride of
2 and the same padding; the second convolution layer has 20 filters and a kernel size of
20; the last convolution layer is composed of 10 filters and a kernel size of 5. Finally, a
fully connected layer is used with a 43 neurons layer and a neuron in the output layer, all
with linear activation functions. Additionally, the batch normalization layer is added with
ReLU activation in various parts of the network. The loss function used was MSE, and the
optimizer was the stochastic gradient descent with momentum. For training, a maximum
of 40 epochs was available with an initial learning rate of 0.001. In this case, 30% of the
data was used to validate, and 70% of the data to train. The parameters of the layers for this network were found in such a way that the lowest
possible MSE will be achieved. Increasing the number of layers, neurons, filter size, or
number of filters did not correspond to a significant improvement performance. p
g
p
p
The second dataset was used for the classification process; it is composed of 4177 data,
of which 3685 data correspond to normal operating conditions, 50 belong to the first attack
scenario, 24 correspond to the second attack scenario, 60 to third and fifth attack, 94 to
fourth and sixth attack, and 110 to the seventh scenario. As can be seen in Figure 6a,
this dataset is unbalanced and would then generate problems to the classifier. The bar
centered at 0 corresponds to normal operating conditions, while the other corresponds
to the different attack scenarios which are shown in the ID column of Table 2. It could
affect the algorithms in relation to the minority classes. 5.1.1. Dataset Description Training Dataset 1 and Training Dataset 2 were used. The first one corresponds to data
collected under normal operating conditions. This dataset was released on November 20,
2016 and was generated from a one-year long simulation. The second dataset corresponds
to situations when attack scenarios are generated. This dataset with partially labeled data
was released on 28 November 2016. The dataset is around six months long and contains
several attacks, as shown in Table 2. Table 2. Attacks featured in Training dataset 2 [78]. ID
Duration (Hours)
Attack Description
SCADA Concealment
1
50
Attackers change L_T7 thresholds (which controls
PU10/PU11) by altering SCADA transmision to
PLC9. Low levels in T7. Replay attack on L_T7. 2
24
Like Attack # 1. Like Attack # 1 but replay attack extended to
PU10/PU11 flow and status. 3
60
Attackers alter L_T1 readings sent by PLC2 to
PLC1, which reads a constant low level and keeps
pumps PU1/PU2 ON. Overflow in T1
Polyline to offset L_T1 increase. Table 2. Attacks featured in Training dataset 2 [78]. 12 of 28 Electronics 2021, 10, 2238 Table 2. Cont. ID
Duration (Hours)
Attack Description
SCADA Concealment
4
94
Like Attack # 3. Replay attack on L_T1, PU1/PU2 flow and
status, as well as pressure at pumps outlet. 5
60
Working speed of PU7 reduced to 0.9 of nominal
speed causes lower later levels in T4. 6
94
Like Attack # 5, but speed reduced to 0.7. L_T4 drop concealed with replay attack. 7
110
Like Attack # 6. Replay attack on L_T1, as well as PU1/PU2
flow and status. Table 2. Cont. Table 2. Cont. 5.1.2. Data Preparation and Model Training For training, a maximum of 4 epochs was available, with an
initial learning rate of 0.0001, and 30% of the dataset was used to validate, while 70% was
used to train. Figure 7. Classification model for SWaT dataset. Figure 7. Classification model for SWaT dataset. The loss function used was CCE, and the optimizer used was stochastic gradient
descent with momentum. For the training, a maximum of 4 epochs was available, with an
initial learning rate of 0.0001. For training, a maximum of 4 epochs was available, with an
initial learning rate of 0.0001, and 30% of the dataset was used to validate, while 70% was
used to train. 5.1.2. Data Preparation and Model Training To address this situation, initially,
methods, such as Random Oversampling and Undersampling, were used for imbalanced
classification without obtaining satisfactory results. For this reason, the approach shown in
Reference [79] was followed. This proposal is a modification of temporal data determined
by optimal sequences that are aligned with the original data, thus generating new time- Electronics 2021, 10, 2238 13 of 28 synthesized data to the training dataset. The distribution of the different classes for the new
dataset to be used is shown in Figure 6b. Although it is observed that it is an unbalanced
dataset, the amount of data generated from the attack scenarios was increased, and the
performance was improved. synthesized data to the training dataset. The distribution of the different classes for the new
dataset to be used is shown in Figure 6b. Although it is observed that it is an unbalanced
dataset, the amount of data generated from the attack scenarios was increased, and the
performance was improved. (a) Original SWAT dataset distribution. (b) New SWAT dataset distribution. Figure 6. SWAT dataset distribution. (a) Original SWAT dataset distribution. (b) New SWAT dataset distribution. Figure 6. SWAT dataset distribution. This new dataset was used to estimate the outputs using the architecture shown in
Figure 5, which were compared with the usual process variables to obtain the residual signal The input data for the classifier whose architecture is shown in Figure 7 are: the
estimated outputs, the process variables, and the corresponding residuals. This corresponds
to the “Classification model” block shown in Figure 2 and was implemented by a group
of cascaded convolutional layers with a batch normalization layer with ReLU activation
function between them. The number of convolutional layers selected was five, obtaining a
higher accuracy than 90%. The number of filters implemented from the input to the fully
connected layer were 128, 64, 32, 16, and 8, respectively. The kernel size in each one was
10. The fully connected layer is composed of eight neurons in its input layer with linear
activation function, while the last layer has eight neurons with softmax activation functions
corresponding to the 7 attacks and the usual operation scenarios. Figure 7. Classification model for SWaT dataset. The loss function used was CCE, and the optimizer used was stochastic gradient
descent with momentum. For the training, a maximum of 4 epochs was available, with an
initial learning rate of 0.0001. 5.1.3. Evaluation Metrics The metrics considered in this work were true positives (TP), false positives (FP),
true negatives (TN), and false negatives (FN). In order to evaluate the performance of
the architecture proposed, the following metrics were used: precision, accuracy, recall Electronics 2021, 10, 2238 14 of 28 14 of 28 (sensitivity or TPR), F1 score, and true negative rate or specificity (TNR). These metrics
were calculated as follows:
TP (sensitivity or TPR), F1 score, and true negative rate or specificity (TNR). These metrics
were calculated as follows:
TP s follows:
Precision =
TP
TP + FP,
(10)
Accuracy =
TP + TN
TP + TN + FP + FN ,
(11)
Recall =
TP
TP + FN ,
(12)
F1 Score =
2TP
2TP + FP + FN = 2 Precision × Recall
Precision + Recall ,
(13)
TNR =
TN
FP + TN . (14) Precision =
TP
TP + FP,
(10)
Accuracy =
TP + TN
TP + TN + FP + FN ,
(11)
Recall =
TP
TP + FN ,
(12)
F1 Score =
2TP
2TP + FP + FN = 2 Precision × Recall
Precision + Recall ,
(13)
TNR =
TN
FP + TN . (14) (10) (11) (12) (13) TNR =
TN
FP + TN . (14) (14) Additionally, the ROC (Receiver Operating Characteristics) and Precision-Recall
Curves were considered. Additionally, the ROC (Receiver Operating Characteristics) and Precision-Recall
Curves were considered. 5.1.4. Analysis of Results of SWaT Case The results obtained for this dataset are shown in this section. The training and
recovering results are carried out in MATLAB software. Figure 8 shows the confusion
matrix for each of the available classes. From these results, the metrics defined in the
previous section are obtained and are presented in Table 3. Figure 8. Confusion matrix for SWaT dataset. Figure 8. Confusion matrix for SWaT dataset. Figure 8. Confusion matrix for SWaT dataset. Table 3. Summary of metrics. Accuracy
Precision
Recall
F1 Score
TNR
Class 0
0.97
0.81
0.97
0.88
0.98
Class 1
0.99
0.99
0.99
0.99
0.99
Class 2
0.99
0.98
0.91
0.94
0.99
Class 3
0.99
0.99
0.95
0.97
0.98
Class 4
0.99
0.94
0.94
0.94
0.99
Class 5
0.99
0.98
0.97
0.97
0.98
Class 6
0.99
0.99
0.99
0.99
0.99
Class 7
0.98
0.95
0.89
0.92
0.98 Electronics 2021, 10, 2238 15 of 28 15 of 28 Class 0 corresponds to the usual operation, while class 1 to 7 are the different attacks
scenarios shown in Table 2. It is observed that accuracy is high in all cases. The above
shows a high percentage ratio of samples correctly classified by our model. On the other
hand, for precision, it is observed that all attack scenarios present a score above 0.94, which
means that a lot of data was correctly classified in the different attack scenarios. Similarly,
the recall scores are above 0.91 in the majority of classes, which allows minimizing the false
alarm rate. Finally, the F1 score shows scores above 0.92. The high rate of TNR in each of
the classes is highlighted, which means that FPR is low. g
g
The ROC and Precision-Recall Curves shown in Figure 9a,b present an appropriate per-
formance, indicating that the model has a good capability to distinguish different classes. (a) ROC curve. (b) Precision-Recall Curve. Figure 9. ROC and Precision-Recall Curves for SWaT dataset. (a) ROC curve. (b) Precision-Recall Curve. Figure 9. ROC and Precision-Recall Curves for SWaT dataset. Table 4 presents a comparison of the proposal presented in this is paper with other
methods. In the recall and F1 score metrics, the proposed method presents a better perfor-
mance related to the other methods. For values of precision and accuracy, the proposed
method is above in almost all cases, except for the last two methods, which exceed it by a
score margin of 0.04. 5.1.4. Analysis of Results of SWaT Case However, the performance of the F1 score metric is high, indicating
that a satisfactory and reliable class detection was obtained. Table 4. Summary of the results and performance comparison on the SWaT dataset. Method
Accuracy
Precision
Recall
F1 Score
Proposed
0.95
0.95
0.95
0.95
SVM [59]
-
0.93
0.70
0.79
RNN [59]
-
0.94
0.70
0.80
1D CNN [60]
-
0.96
0.80
0.87
TABOR [61]
0.95
0.86
0.79
0.82
STAE-AD [63]
-
0.96
0.82
0.88
AE [64]
-
0.89
0.80
0.84
AE Frequency [64]
-
0.92
0.83
0.87
LSTM [62]
-
0.98
0.68
0.88
One Class SVM [62]
-
0.93
0.70
0.80
SDA+1D CNN+ LSTM [42]
0.99
0.99
0.85
0.91
SDA+1D CNN+ GRU [42]
0.99
0.99
0.85
0.92
5 2 Interconnected Tank Testbed Table 4. Summary of the results and performance comparison on the SWaT dataset. 5.2. Interconnected Tank Testbed 5.2. Interconnected Tank Testbed This testbed has been used extensively to test proposals to detect anomalies [37,65–69]. The hydraulic system consists of three identical cylindrical tanks with equal cross-sectional
area S, as shown in Figure 10. These tanks are connected by two cylindrical pipes of the
same cross-sectional area, denoted Sn, and have the same outflow coefficient, denoted µ13
and µ32. The nominal outflow located at tank 2 has the same cross-sectional area as the
coupling pipe between the cylinders, but a different outflow coefficient, denoted µ20. The Electronics 2021, 10, 2238 16 of 28 16 of 28 inlet flow of the tanks comes from two pumps, with a flow rate, q1 and q2. A digital/analog
converter is used to control each pump. A piezo-resistive differential pressure sensor
carries out the necessary level measurement. The idea of the system is to maintain the
height levels of the fluid stored in tanks 1 and 2 at a particular operating point. Figure 10 Schematic diagram of the three tank system Figure 10. Schematic diagram of the three-tank system. The parameters are shown in Table 5, and the mathematical model is presented in
(15)–(17) [58]. dl1(t)
dt
= (q1(t) −q13(t))/S
dl2(t)
dt
= (q2(t) + q32(t) −q20(t))/S
dl3(t)
dt
= (q13(t) −q32(t))/S
,
(15) (15) qmn(t) = µmnSpsign(lm(t) −ln(t))
q
2g|lm(t) −ln(t)| (m, n = 1, 2, 3 ∀m ̸= n),
(16)
q20(t) = µ20Sp
q
2gl2(t). (17) qmn(t) = µmnSpsign(lm(t) −ln(t))
q
2g|lm(t) −ln(t)| (m, n = 1, 2, 3 ∀m ̸= n),
(16)
q20(t) = µ20Sp
q
2gl2(t). (17) (16) (17) Table 5. Parameters value of the three-tank system. Table 5. Parameters value of the three-tank system. y
Variable
Symbol
Value
Tank cross sectional area
S
0.0154 m2
Inter tank cross sectional area
Sn
5 × 10−5 m2
Outflow coefficient
µ13 = µ32
0.05
Outflow coefficient
µ20
0.675
Maximum flow rate
qimax(i ∈[1 2])
1.2 × 10−4 m3/s
Maximum level
ljmax(j ∈[1 2 3])
0.62 5.2.1. Dataset Generation 5.2.1. Dataset Generation Assuming that l1 > l2 > l3, a linear approximation was established around an
equilibrium point (U0, Y0) using Taylor series expansion. The linearized system is described
by a discrete state space representation with a sampling period of Ts = 1s. This is shown
in (18). x(k + 1) = Ax(k) + Bu(k)
y(k) = Cx(k)
. (18) (18) The states x(k) correspond to the fluid level of the tanks. The states x(k) correspond to the fluid level of the tanks. Electronics 2021, 10, 2238 17 of 28 17 of 28 The purpose of this study is to control system around the operating point (U0, Y0), as
is shown in (19). Y0 = [0.4 0.2 0.3]T(m)
U0 = [0.35 × 10−4 0.375 × 10−4]T(m3/s)
. (19) (19) A tracking control problem was considered in this study case, where the desired
outputs y = [l1 l2]T are required to track references. The state feedback pole assignment
technique was used. Thus, a feedback gain matrix K was designed, so that the closed-loop
eigenvalues of the augmented system are equal to [0.92 0.97 0.9 0.95 0.94]. MATLAB
software was used to find the matrices A and B, as well as the controller gains. The values
can be observed in (20)–(22). A =
0.9888
0.0001
0.0112
0.0001
0.9781
0.0111
0.0112
0.0111
0.9776
,
(20)
B =
64.5687
0.0014
0.0014
64.2202
0.3650
0.3637
,
(21)
K = [K1 |K2] = 10−4
21.6
3
−5
2.9
19
−4
|
−0.95
−0.32
−0.3
−0.91
.
(22) (20) (21) (22) In order to construct the dataset for detecting attacks, the scheme shown in Figure 11
was implemented, which has modules to obtain measurements of the process variables,
as well as the control actions applied by the actuators. An Ethernet was used as a control
network. This representation is equivalent to boxes “Process” and “Controller” in the
architectures presented in Figure 3. Figure 11. Interconnected tank testbed. Figure 11. Interconnected tank testbed. Electronics 2021, 10, 2238 18 of 28 Two datasets were generated. The first one is a dataset in normal operations to
determine a model that estimates the system outputs. The second one includes cyber
attacks on sensors 1 and 2. These cyber attacks can be integrity or DoS attacks. y
g
y
In both cases, 499,000 samples were generated. 5.2.1. Dataset Generation The system references range between
0.35 m and 0.45 m for l1, and between 0.185 m and 0.25 m for l2. The time intervals were
defined randomly with a uniform distribution and reference changes every 500 s to 850 s. The cases are shown in Table 6. Case 0 corresponds to operation without attacks. The
following cases correspond to situations in which integrity or DoS cyber attacks can be
generated on any sensor, following the models described by the Equations (3) and (4). In
cases 1 to 4, only one cyber attack is generated every time, while cases 5 to 8 correspond to
simultaneous attacks. Table 6. Cases raised. Case
Description
Case 0
Normal operation
Case 1
Integrity attack on sensor 1
Case 2
Integrity attack on sensor 2
Case 3
DoS attack on sensor 1
Case 4
DoS attack on sensor 2
Case 5
Integrity attack on sensor 1 and DoS on sensor 2
Case 6
Integrity attack on sensor 2 and DoS on sensor 1
Case 7
Integrity attack on sensor 1 and 2
Case 8
DoS attack on sensor 1 and 2 Table 6. Cases raised. Table 6. Cases raised. The time intervals in which cyber attacks occur were defined such that the dataset was
balanced, so it were defined randomly and uniformly distributed. The integrity attacks
were implemented by changing the modified variable in a range of 5% to 8% of its measured
value. This range of values depends on the sensitivity of the system since there will be
particular processes where the effect of the variation of the measurements in a given range
does not has as much impact as in others. All cases presented correspond to the classes
that the classifier will identify. The distribution of these data is shown in Figure 12. Figure 12. Dataset for cyber attack classification. Figure 12. Dataset for cyber attack classification. Figure 12. Dataset for cyber attack classification. 5.2.2. Model Training 5.2.2. Model Training Figure 3 presents the architecture implemented. The first model generates the process
states estimate, while two more models were obtained to reconstruct independent states x1
and x2, according to those states susceptible to cyber attack. Electronics 2021, 10, 2238 19 of 28 The first network has the architecture shown in Figure 13. Its input data is composed
of five characteristics, which are composed of the measurements of the sensors and the
control actions corresponding to vector (23): input data =[x1(1), . . . , x1(k −1), x2(1), . . . , x2(k −1), x3(1), . . . , x3(k −1),
u1(1), . . . , u1(k −1), u2(1), . . . , u2(k −1)]T
. (23) (23) 1( ),
,
1(
),
2( ),
,
2(
)]
Figure 13. Model to estimate all states. Figure 13. Model to estimate all states. The model has three outputs corresponding to the states of the process. The vector to
be reconstructed is (24): output data 1 = ˆx1 = [x1(2), . . . , x1(k)]T
output data 2 = ˆx2 = [x2(2), . . . , x2(k)]T
output data 3 = ˆx3 = [x3(2), . . . , x3(k)]T
,
(24) (24) where k is the number of samples. This model has two convolutional layers, one average
pooling 1D layer between the convolutional layers, and one fully connected layer. The first
convolutional layer has a kernel size of 5 and has eight filters, while the second layer has
a kernel size of 3 with 16 filters. Each of these layers has hyperbolic tangent activation
function. Between previous layers, there is an average pooling 1D layer with a pool size
of 2 and strides of 2 with same padding. Between the convolutional layers and the fully
connected layer, there is a batch normalization layer with Leaky ReLU type activation
function. In the fully connected layer, there is an input layer of 48 neurons and an output
layer composed of 3 neurons with a linear activation function to estimate the corresponding
states. The loss function used was MSE, and the optimizer used was Adam. For training, a
maximum of 4 epochs and a batch size of 10 were available with initial learning rate of 0.01. To train the model, 30% of the data was used to validate, and 70% to train. 5.2.2. Model Training The various
parameters of the layers of this network were found in such a way that the lowest possible
MSE will be achieved, it was 0.000067. Increasing the number of layers, neurons, filter
size, or number of filters did not correspond to a significant improvement to the proposed
architecture. The second and third networks have the architecture shown in the Figure 14. Figure 14. Model to estimate the decoupled states. Figure 14. Model to estimate the decoupled states. Electronics 2021, 10, 2238 20 of 28 The input data for the second architecture is composed of four characteristics corre-
sponding to the measurements of the sensors and the control actions, as is presented in
vector (25): input data =[x2(1), . . . , x2(k −1), x3(1), . . . , x3(k −1),
u1(1), . . . , u1(k −1), u2(1), . . . , u2(k −1)]T. (25) (25) The model generates an estimated uncoupled output for the first state as (26): The model generates an estimated uncoupled output for the first state as (26): output data = ˆx1d = [x1(2), . . . , x1(k)]T,
(26) (26) output data = ˆx1d = [x1(2), . . . , x1(k)]T, where k is the number of samples. This model has two convolutional layers and one fully
connected layer. The first convolutional layer has a kernel size of 4 and has 8 filters, while
the second layer has a kernel size of 2 with 16 filters. Each of these layers has the hyperbolic
tangent activation function. Between these layers, there is an average pooling 1D layer
with a pool size of 2 and a stride of 2 with the same padding. Between the convolutional
layers and the fully connected layer, there is a batch normalization layer and a Leaky
ReLU type activation function. Before the fully connected layer, a dropout layer (0.15) was
added. In the fully connected layer, there is an input layer of 32 neurons and an output
layer composed of 1 neuron with linear activation function to estimate the corresponding
state. The loss functions used was MSE, and the optimizer used was Adam. For training, a
maximum of 4 epochs and a batch size of 10 was available with initial learn rate of 0.01. Seventy percent of the data was used to train the model, and 30% to validate it. 5.2.2. Model Training The various
parameters of the layers of this network were found in such a way that the lowest possible
MSE will be achieved, and it was 0.00047. Increasing the number of layers, neurons, filter
size, or number of filters did not correspond to a significant improvement to the proposed
architecture. Finally, the structure used to estimate the second uncoupled state of the process is
shown in (27) and (28). The respective MSE for this case was 0.000031. input data =[x1(1), . . . , x1(k −1), x3(1), . . . , x3(k −1),
u1(1), . . . , u1(k −1), u2(1), . . . , u2(k −1)]T,
(27) (27) output data = ˆx2d = [x2(2), . . . , x2(k)]T
(28) output data = ˆx2d = [x2(2), . . . , x2(k)]T (28) The architecture proposed for the classifier of the cyber attack is similar to that shown
in Figure 7. It is composed of three convolutional layers whose activation function is
hyperbolic tangent. The first convolutional layer has a kernel size of 15 with several
80 filters. The second and third convolutional layers have the same kernel size, but the
number of filters is 60 and 30, respectively. There is also a batch normalization layer with
Leaky ReLU activation function. Finally, a fully connected layer is used with an input layer
of 25 neurons and an output layer with nine neurons corresponding to the established
classes above. The last layer uses the softmax function. The loss function used was CCE,
and the optimizer used was stochastic gradient descent with momentum. For training, a
maximum of 1000 epochs was established, with a batch size of 10 and initial learning rate
of 0.0001. For model training, 30% of the data was used to validate, and 70% to train. 5.2.2. Model Training The
input data is (29): (29) input data = [x1, x2, x3, ˆx1, ˆx2, ˆx3, ˆx1d, ˆx2d, q1, q2, res, res1, res2]T,
(29) where x1, x2, x3 correspond to the real variables of the process; ˆx1, ˆx2, ˆx3 are the outputs
estimated by the architecture shown in Figure 13; ˆx1d and ˆx2d correspond to the decoupled
states estimated by the architecture of the Figure 14, q1 and q2 are the process references,
and res, res1, and res2 are the residual signals obtained by comparing the real process states
with the estimated states, and the individual comparison between the first two real process
states and the estimated decoupled states, respectively. Electronics 2021, 10, 2238 21 of 28 21 of 28 Figure 15a,b present the evolution of the cost function and the accuracy metric obtained
during the classifier training procedure. (a) Accuracy. (b) Loss function. Figure 15. Training and Validation: accuracy and loss function through epochs. (a) Accuracy. (a) Accuracy. (b) Loss function. y
gure 15. Training and Validation: accuracy and loss function through epochs. Figure 15. Training and Validation: accuracy and loss function through epochs. Python programming language and the Keras library were used for training and
obtaining the results. With the purpose of evaluating the performance of this architecture,
the same metrics of the previous case were used. 5.2.3. Performance Analysis of the Three-Tank System Testbed Indices values obtained for this case are presented in Figures 16 and 17a,b and Table 7. For accuracy, the best scores were obtained when simultaneous attacks happened with
values above 0.97. The above is an important result because this situation has been little
explored. In terms of recall, class 7 has a slightly fair score, while the other situations have
scores above 0.83. Additionally, the F1 score also has high values. The scores show that the
proposed architecture allows for high specificity and high sensitivity. Figure 16. Confusion matrix for the three-tank system. Figure 16. Confusion matrix for the three-tank system. 22 of 28 Electronics 2021, 10, 2238 (a) ROC Curve. (b) Precision-Recall Curve. Figure 17. ROC and Precision-Recall Curves for Interconnected tank. (a) ROC Curve. (b) Precision-Recall Curve. Figure 17. ROC and Precision-Recall Curves for Interconnected tank. Figure 17. ROC and Precision-Recall Curves for Interconnected tank. y
Accuracy
Precision
Recall
F1 Score
TNR
Class 0
0.97
0.83
0.96
0.89
0.98
Class 1
0.96
0.89
0.83
0.86
0.99
Class 2
0.97
0.89
0.84
0.87
0.99
Class 3
0.98
0.86
0.97
0.91
0.98
Class 4
0.98
0.87
0.96
0.91
0.98
Class 5
0.98
0.91
0.86
0.89
0.99
Class 6
0.98
0.92
0.88
0.90
0.99
Class 7
0.96
0.94
0.72
0.81
0.99
Class 8
0.99
0.94
0.99
0.97
0.99 The alarm indicator was implemented from the classifier in order to know the process
state. Since the classifier provides the probability to classify an input data in particular
class, the alarm signal is generated taking in to account the maximum value obtained from
the classifier. In Figure 18, the alarm indicator is 1 when sensor 1 or 2 is under attack, and 0
when it is not. Additionally, it is discriminated if the attack is DoS or integrity type. The
response of the process when it is attacked is shown in Figure 19. Boxes indicate the time
instance when the attack occurs in both sensors, according to the alarm signals generated. Red boxes correspond to DoS attacks and black boxes correspond to integrity attacks g
g
g
Red boxes correspond to DoS attacks, and black boxes correspond to integrity attacks. Additionally, the effect is different, depending on whether it is DoS or integrity
attack. The system proposed in this work performed appropriately to detect the occurrence
of the cyber attack, as well as the location and type of the attack. 5.2.3. Performance Analysis of the Three-Tank System Testbed As results obtained
using convolutional networks were better than those employing RNN or LSTM networks,
convolutional networks were then chosen for this proposal. p
p
In summary, the key steps for using the proposed architecture are as follows: p
p
In summary, the key steps for using the proposed architecture are as follows: 1. Generate an estimated output of the process under a regression model. 2. Generate a residual signal under the comparison of the measured process outputs
with estimated outputs. 3. Use a classification model that from some system characteristics, such as control
actions, estimated outputs, measured process outputs, and residual signals, allows
evaluating if there is an attack in any part of the system. g
y p
y
4. From the detected class, generate alarm signals to report the occurrence of a cyber
attack to define the type of attack and the part of the system that is being affected
by it. 23 of 28 Electronics 2021, 10, 2238 Figure 18. Alarm generation. Figure 19. Temporal response. 6. Conclusions
New applications of industrial automation request great flexibility in the systems
which is supported by the increase in the interconnection between its components. At the
same time, it generates a large gap that affects the security of control systems. Current
solutions are oriented mainly to avoid the occurrence of attacks, but, regardless, the
problems appear; so, recently, the interest in developing new proposals that contribute to
d t
t tt
k h Figure 18. Alarm generation. Figure 18. Alarm generation. Figure 18. Alarm generation. Figure 19. Temporal response. Figure 19. Temporal response. 6. Conclusions New applications of industrial automation request great flexibility in the systems,
which is supported by the increase in the interconnection between its components. At the
same time, it generates a large gap that affects the security of control systems. Current
solutions are oriented mainly to avoid the occurrence of attacks, but, regardless, the
problems appear; so, recently, the interest in developing new proposals that contribute to
detect attacks has grown. In this work, a new architecture for DoS and integrity cyber attacks detection and iso-
lation in Cyber Physical Systems using one-dimensional Convolutional Neural Networks
was presented, thereby overcoming other models that are based on machine learning and Electronics 2021, 10, 2238 24 of 28 24 of 28 model-based methods, such as the use of Unknown Input Observers. This architecture
involves a series of steps to achieve its purpose. The first step was to generate an estimated
output of the process under a regression model. The next step was to generate a residual
signal under the comparison of the measured process outputs with estimated outputs. Then, a classification model was added whose input data are different characteristics, such
as control actions, estimated outputs, measured process outputs, and residual signals. This
model allowed for detection and isolation of different eventualities that were defined in
classes. Finally, from the detected class, alarm signals were generated that are used to
report the occurrence of a cyber attack, allowing to define the type of attack and the part of
the system that is being affected by the attack. The architecture proposed does not use threshold information to detect and isolate
attacks, as is the case with model-based methods, such as Unknown Input Observers, which
often use this information. These models require an exhaustive selection of these thresholds,
which can cause both false detections and anomalous situations that go undetected, and
the proposed architecture provides shows advantages over this. p
p
p
g
The performance of the proposed architecture was validated by two test benches
obtaining satisfactory results compared to other methods. The results on the SWaT dataset
allowed observing that, in terms of precision and accuracy, the indexes are very close to
the highest scores of other works, and these obtained a score of 0.95. In terms of recall and
F1Score metrics, it presented a score of 0.95, which outperforms the previously proposed
methods by a good margin. 6. Conclusions Overall, the proposed system has a high true positive rate and
a low false positive rate. On the other hand, the ability of the system to be able to detect and
isolate cyber attacks that may occur simultaneously is highlighted, which was presented in
the three-tank system testbed. In the defined classes, the accuracy presents scores above
0.96, and the precision is above 0.83, in cases where attacks occur in a single part of the
system, while the score is higher than 0.91 in cases where simultaneous attacks occur. In
terms of the F1 score metric, the scores are above 0.81, which is a very promising result. Finally, with respect to the recall metric, the scores are above 0.83, in most cases. With the
cases presented in this testbed, it was possible to demonstrate the ability of the proposed
architecture to detect and locate attacks that may occur simultaneously. This is interesting
because these types of experiments are rarely performed, let alone provide evidence of
systems that can detect these types of situations, which are not alien to eventualities that
may occur in reality. In both cases highlighted, there was a high rate of TNR in each of the
classes, ranging between 0.98 and 0.99. Author Contributions: Conceptualization, C.M.P.; methodology, C.M.P. and D.M.-C.; software,
C.M.P.; validation, C.M.P. and D.M.-C.; formal analysis, C.M.P.; investigation C.M.P. ; resources, D.M.-
C.; data curation, C.M.P.; writing—original draft preparation, C.M.P.; writing—review and editing,
C.M.P, D.M.-C., V.I.-J. and A.G.-P.; visualization, D.M.-C. and A.G.-P; supervision, D.M.-C.; project
administration, D.M.-C. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding Funding: This research received no external funding. Funding: This research received no external funding. Data Availability Statement: Interested partis can contact the first author about the availability
of datasets. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations Abbreviations
The following abbreviations have been used in this manuscript:
AE
Autoencoder
CCE
Categorical crossentropy
CPS
Cyber Physical System
CNN
Convolutional Neural Network
DBN
Deep Belief Networks
DoS
Denial of Service The following abbreviations have been used in this manuscript:
AE
Autoencoder
CCE
Categorical crossentropy
CPS
Cyber Physical System
CNN
Convolutional Neural Network
DBN
Deep Belief Networks
DoS
Denial of Service 25 of 28 Electronics 2021, 10, 2238 FNR
False negative rate
FPR
False positive rate
GRU
Gated recurrent unit
ICS
Industrial Control System
IDS
Intrusion Detection System
LO
Luenberger Observer
LSTM
Long short-term memory
MSE
Mean squared error
PCA
Principal Component Analysis
PLC
Programmable Logic Controller
ROC
Receiver Operating Characteristics
RNN
Recurrent Neural Network
SDA
Stacked denoising autoencoder
STAE-AD
Spatio-Temporal Autoencoder for Anomaly Detection
SVM
Support Vector machine
SWaT
Secure Water Treatment
TNR
True negative rate
TPR
True positive rate
UIO
Unknown Input Observer FNR
False negative rate
FPR
False positive rate
GRU
Gated recurrent unit
ICS
Industrial Control System
IDS
Intrusion Detection System
LO
Luenberger Observer
LSTM
Long short-term memory
MSE
Mean squared error
PCA
Principal Component Analysis
PLC
Programmable Logic Controller
ROC
Receiver Operating Characteristics
RNN
Recurrent Neural Network
SDA
Stacked denoising autoencoder
STAE-AD
Spatio-Temporal Autoencoder for Anomaly Detection
SVM
Support Vector machine
SWaT
Secure Water Treatment
TNR
True negative rate
TPR
True positive rate
UIO
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An Evaluation for Model Testability approaches
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International journal of computer and technology
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cc-by
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An Evaluation for Model Testability approaches
Pourya Nikfard1, Suhaimi bin Ibrahim2, Babak Darvish Rohani3, Harihodin bin Selamat4, Mohd
Naz‟ri Mahrin5
Advanced Informatics School (AIS), University Technology Malaysia (UTM), International campus, Kuala Lumpur, An Evaluation for Model Testability approaches
Pourya Nikfard1, Suhaimi bin Ibrahim2, Babak Darvish Rohani3, Harihodin bin Selamat4, Mohd
Naz‟ri Mahrin5
Advanced Informatics School (AIS), University Technology Malaysia (UTM), International campus, Kuala Lumpur,
Malaysia
{1npourya2@live.utm.my, 2suhaimiibrahim@utm.my, 3drbabak3@live.utm.my,
4h
ih di @i
t
5
d
i
@ t
} ISSN 22773061 ISSN 22773061 1. Introduction Either they do (1) not think about the context of models of software,
(2) not propose a systematic process for choosing and developing right dimensions, (3) not describe a reliable and general understanding of quality, or (4) not separate between subjective and objective
measurements (Voigt & Engels, 2008). Testability is a property of program that is introduced with the purpose of forecasting efforts need for testing the program. To
quantify the program testability is to relate the program with some numbers to present the degree of how easy are those
definite testable properties in the program to be tested. High testability software means that errors could be discovered more
easily during testing (if the software exist errors). Otherwise, it is complicated to be tested and is likely to be fewer reliable. Therefore the number of tests need in a program may be taken as the quantify of its testability. There has not been an
instinctively acceptable measure of program testability. Software testability is the degree to which a software artifact (i.e. a
software module, software system, design document or requirements) supports testing in a test context that given. Testability
is not an inherent property of a software artifact and cannot be calculated directly (such as software size). Instead testability is
an extrinsic property that results from software interdependency to be tested and the test goals, test methods used, and test
resources (i.e., the test context). A minor degree of testability results in enhanced test effort. In tremendous cases a testability
lack may hinder software testing parts or software requirements at all (Yeh & Lin, 1998). 1. Introduction Software testing is a significant part of both the software lifecycle and quality assurance activities. In simple form, testing is the
software products dynamic execution in order to show the errors presence. Software testing is costly, and it is considered as
the final barrier to the software product release. Model-based testing (MBT) is an automation of black box testing technique; its
major difference from the conventional methods of black box testing is that the test cases are automatically produced by
software tools that develop the expected behavioral models of the software under the test (SUT). MBT has conveyed a lot of benefits. These contain errors early detection, which is measured to be very cheap. The major MBT liability
is to knowing how to model SUT (Reza, Ogaard, & Malge, 2008). Model-based testing (MBT) is a rising trend in test automation. In MBT, the SUT is modeled at an appropriate level of
abstraction for testing, and tools are used to automatically create test cases based on this model. Given a set of appropriate
methods and tools, MBT has been demonstrated to be a helpful and efficient means for high-level testing in diverse domains. Some benefits comprise decreased costs of maintenance in concentrating on a single high-level model, and boosted test
coverage over the features expressed in the test model by the means of automated test generation (Puolitaival & Kanstrén,
2010). Model-based testing (MBT) defendants the systematic use of software models in particular for testing activities, e.g. creation of
test oracles, test execution environments and test cases. If a software model is well appropriate for activities of testing, we
speak of a testable model. Thus, software models testability is a significant quality representing the degree to which a software
model helps activities of testing in a given test context. The software models testability should already be analyzed and
enhanced at modeling time before MBT activities start. This assists to save costs for the detection and correction of defects of
testability in later stages. For models used in model-based testing, their testability evaluation is a significant problem. Existing approaches lack some
related features for a systematic and complete evaluation. 2.2 Analysis of Object Oriented Complexity and Testability Using Object Oriented
D
i
M t i 2.2 Analysis of Object Oriented Complexity and Testability Using Object Oriented
Design Metrics 2.1 An Empirical Analysis of a Testability Model for Object-Oriented Authors presented a metric based testability model for object-oriented programs. The paper investigated empirically the
relationship between the model and testability of classes at the code level. Testability has been investigated from the
perspective of unit testing. They designed an empirical study using data collected from two open sources Java software
systems for which JUnit test cases exist. To capture testability of classes, they used various metrics to quantify different
characteristics of the corresponding JUnit test cases. In order to evaluate the capability of the model to predict testability of
classes, they used statistical tests using correlation. The achieved results support the idea that there is a statistically
significant relationship between the model and the used test case metrics (Kout et al., 2011). An Evaluation for Model Testability approaches
Pourya Nikfard1, Suhaimi bin Ibrahim2, Babak Darvish Rohani3, Harihodin bin Selamat4, Mohd
Naz‟ri Mahrin5
Advanced Informatics School (AIS), University Technology Malaysia (UTM), International campus, Kuala Lumpur,
Malaysia
{1npourya2@live.utm.my, 2suhaimiibrahim@utm.my, 3drbabak3@live.utm.my,
4harihodin@ic.utm.my, 5mdnazrim@utm.my} {1npourya2@live.utm.my, 2suhaimiibrahim@utm.my, 3drbabak3@live.utm.my,
4harihodin@ic.utm.my, 5mdnazrim@utm.my} {1npourya2@live.utm.my, 2suhaimiibrahim@utm.my, 3drbabak3@live.utm.my,
4harihodin@ic.utm.my, 5mdnazrim@utm.my} Abstract- Design for testability is a very important issue in software engineering. It becomes crucial in the case of Model
Based Testing where models are generally not tested before using as input of Model Based Testing. The quality of design
models (e.g.; UML models), has received less attention, which are main artifacts of any software design. Testability tends to
make the validation phase more efficient in exposing faults during testing, and consequently to increase quality of the end-
product to meet required specifications. Testability modeling has been researched for many years. Unfortunately, the modeling
of a design for testability is often performed after the design is complete. This limits the functional use of the testability model
to determining what level of test coverage is available in the design. This information may be useful to help assess whether a
product meets the target requirement to achieve a desired level of test coverage, but has little proactive effect on making the
design more testable. product meets the target requirement to achieve a desired level of test coverage, but has little proactive effect on making the
design more testable. Council for Innovative Research
Peer Review Research Publishing System
Journal: INTERNATIONAL JOURNAL OF COMPUTERS & TECHNOLOGY
Vol 9, No 1 Council for Innovative Research
Peer Review Research Publishing System
Journal: INTERNATIONAL JOURNAL OF COMPUTERS & TECHNOLOGY
Vol 9, No 1 Vol 9, No 1 editor@cirworld.com
www.cirworld.com, member.cirworld.com editor@cirworld.com
www.cirworld.com, member.cirworld.com J u l y 1 5 , 2 0 1 3 J u l y 1 5 , 2 0 1 3 938 | P a g e ISSN 22773061 2.3 Metric Based Testability Model for Object Oriented Design (MT The proposed model for the assessment of testability in object-oriented design has been validated using structural and
functional information from object oriented software. The models‟ ability to estimate overall testability from design information
has also been demonstrated using several functionally equivalent projects where the overall testability estimate computed by
model had statistically significant correlation with the assessment of overall project characteristics determined by independent
evaluators. The proposed model is more practical in nature having quantitative data on testability is of immediate use in the
software development process. The software developer can use such data to plan and monitor testing activities. The tester
can use testability information to determine on what module to focus during testing. And finally, the software developer can
use testability metrics to review code, trying to find refactoring that would improve the testability of the code (Khan & Mustafa,
2009). 2.4 Automating regression test selection based on UML designs The authors propose a methodology supported by a prototype tool to tackle the regression test selection problem at the
architecture/ design level in the context of UML-based development. Their main motivation is to enable, in the context of UML-
based development, regression test selection based on design change information, early in the change process. They also present three case studies that were used as an initial feasibility and benefit assessment. These case studies are varied
in the sense that they cover very different systems and changes, in both industrial and academic settings. Results show that
design changes can have a complex impact on regression test selection and that, in many cases, automation is likely to help
avoid human errors. Their objective has been to ensure that regression testing was safe while minimizing regression testing
effort. But they have shown that certain changes may not be visible in the design and may require additional attention during
coding or a special way to document them during design. Another limitation is that, based on UML design information, test
selection may not be as precise as if it was based on detailed code analysis. Improving precision by analyzing in more detail
guard conditions and OCL contracts is the focus of their current research. However, their case studies have only shown one
case of imprecision in classifying a test case as retestable. Despite the above limitations, by providing a methodology and tool
to perform impact analysis and regression test selection based on UML designs, the achievements are: – Higher efficiency in test selection based on the automation of design change analysis and traceability between UML designs
and regression test cases. Higher efficiency in test selection based on the automation of design change analysis and traceabili
nd regression test cases. – Better support for assessing and planning regression test effort earlier in the change process that is once design changes
have been determined (Briand et al., 2009). Better support for assessing and planning regression test effort earlier in the change process that
ave been determined (Briand et al., 2009). 2.5 Automatic generation of test specifications for coverage of system state
transitions The authors have presented a novel strategy for automatic state-based system testing for achieving transition path coverage. A severe handicap in system test generation for transition coverage arises due to the fact that system state models are not
developed during any pragmatic development process. This may be attributed to the fact that the state models for practical
problems tend to be extremely large and complex. To overcome this problem, they synthesize a system state model of an
object-oriented system from the relevant UML models. The synthesized state model is then used to generate test
specifications for transition coverage. Their approach to system testing does not replace traditional testing approaches such
as, method coverage, message path coverage, etc. On the other hand, their approach to testing based on transition path
coverage is intended to be used in a complementary manner with traditional test techniques. The test specification generated
by our approach could detect bugs not detected using traditional testing, since the fault models for the two are different (Sarma
& Mall, 2009). ISSN 22773061 model to the actual software projects. Literature survey concepts and estimated results obtained by actual projects verify each
other. Improvements can be made to the proposed metrics. As more detailed designs are produced during later design
phases, the proposed metrics can be applied to different other types of diagrams produced as a part of detailed design either
in its existing form or by model to the actual software projects. Literature survey concepts and estimated results obtained by actual projects verify each
other. Improvements can be made to the proposed metrics. As more detailed designs are produced during later design
phases, the proposed metrics can be applied to different other types of diagrams produced as a part of detailed design either
in its existing form or by modifying it according to requirements. Complexity analysis at coding stage can be performed by the object oriented design
metrics proposed in this paper (Khalid et al., 2010). 2.2 Analysis of Object Oriented Complexity and Testability Using Object Oriented
Design Metrics The approach proposed in this paper revolves around the complexity and testability of object oriented design. Predicting
complexity of design at class level helps to simplify the design as much as possible. Object oriented design metrics extended
by the approach proposed in this paper helps to obtain the quantifiable results so that complexity of design can be predicted
accurately. The approach is based on literature survey and the concepts developed are validated by mapping the metrics J u l y 1 5 , 2 0 1 3 939 | P a g e 939 | P a g e 939 | ISSN 22773061 2.7 Construction of a Systemic Quality Model for Evaluating a Software Product In referring to the well-known expression “build quality into software,” Dromey points out that high-level quality attributes, such
as reliability and maintainability, cannot be built into the software. What can be done though is to identify a set of properties
(such as modules without side effects) and build them up consistently, harmoniously and fully to provide reliability and
maintainability. Links must be forged between the tangible properties of the product and the high-level quality attributes. Dromey proposes three models, depending on the products resulting from each stage of the development process:
requirements model, design model, and implementation quality model (programming). In comparing this model to ISO 9126,
additional characteristics like process maturity and reusability are noticeable. It is important to point out the weighting Dromey
gives to process maturity, an aspect not considered in previous models. This model seeks to increase understanding of the
relationship between the attributes (characteristics) and the subattributes (subcharacteristics) of quality. It also attempts to
pinpoint the properties of the software product that affect the attributes of quality. After analyzing various product quality
models, the different quality attributes or characteristics found in each of them must be compared. Table 2.9 shows that the
quality characteristics found in the majority of the models are: efficiency, reliability, maintainability, portability, usability and
functionality, which have been present in more recent models. Because they are present in all the models studied, they can be
considered essential and worthy of study (Ortega & Rojas, 2003). ISSN 22773061 ISSN 22773061 by 50% to test a class. Hence, the software practitioners can make use of software contracts to reduce the testing effort and
hence improve the testability of the software (Singh & Saha, 2010). by 50% to test a class. Hence, the software practitioners can make use of software contracts to reduce the testing effort and
hence improve the testability of the software (Singh & Saha, 2010). 2.8 An empirical study into class testability Testability The ISO defines testability as„„attributes of software that bear on the effort needed to validate the software
product‟‟. Binder offers an analysis of the various factors that contribute to a system‟s testability, which he visualizes using the
fish bone diagram as shown in below Figure. The major factors determining test effort Binder distinguishes include the testing
criterion that is required, the usefulness of the documentation, the quality of the implementation, the reusability and structure of
the test suite, the suitability of the test tools used, and the process capabilities. Of these factors, our study is concerned with
the structure of the implementation, and on source code factors in particular. We distinguish between two categories of source
code factors: factors that influence the number of test cases required testing the system, and factors that influence the effort
required to develop each individual test case. We will refer to the former category as test case generation factors and to the
latter category as test case construction factors, which both are discussed below (Bruntink & Van Deursen, 2006). 2.6
Improving the Testability of Object Oriented Software through Software
Contracts The main goal of the study was to establish the relationship between software testability and software contracts. The software
contracts reduce the testing effort and hence improve the testability of an object oriented class. The same is demonstrated
through an example of a queue class written in C++. The results show that software contracts reduce the number of test cases J u l y 1 5 , 2 0 1 3 940 | P a g e 940 | P a g e 940 2.9 Contract-Based Software Component Testing with UML Models This research takes a different approach by incorporating testing-support artefacts (called test contracts) at the model-based
specification level to improve model-based component testability with model-based test contracts. They believe that model-
based testability stands at a testing level above traditional code-based testability, and thus is consistent with and supports
model-based approaches to SCT (Zheng & Bundell, 2008). model based approaches to SCT (Zheng & Bundell, 2008). J u l y 1 5 , 2 0 1 3 941 | P a g e 941 | P a g e ISSN 22773061 2.10 Quality Assessment and Quality Improvement for UML Models The ISO/IEC 9126-model defines no uses, but distinguishes between internal quality, external quality and quality-in-use. The
quality ISO/IEC 9126-model is a generic quality model that covers internal and external quality in one abstract model. The
model for quality-in-use is similar to the operation use of the McCall model. However, quality-in-use and external quality are
out of the scope of this paper, and therefore not discussed any further (Jalbani, 2011). J u l y 1 5 , 2 0 1 3 942 | P a g e 942 | P a g e ISSN 22773061 3. Comparative evaluation In this section we evaluate the above model testability approaches. We declare the achieveme
approach. In this section we evaluate the above model testability approaches. We declare the achievement and main issue of each
approach. 943 | P a g e
J u l y 1
Ro
Authors
w
Method
Achievements
Main Issue
Year
1)investigate empirically the relationship
1)Accountability 2)Accessibility
between the model and testability of classes 3)Communicativeness 4)Not
at the code level
sufficient for Self-Descriptiveness
et al. 2)Design an empirical study using JUnit
5)Not sufficient for both structural
1
2011
UML
3)evaluate the capability of the model to
and behavioural architecture
Kout
predict testability of classes with using
statistical tests 4)using correlation
existing statistically significant relationship
between the model and the used test case
metrics
et
1)Extend the object oriented design metrics
1)Accountability 2)Accessibility
2)obtain the quantifiable results
3)Not sufficient for Self-
Khalid
2
2010
UML
3)predict complexity of design accurately
Descriptiveness
4)Not sufficient for both structural
and behavioural architecture
1)Validate model using structural and
1)Accountability 2)Accessibility
functional information
3)Communicativeness 4)Not
2)Demonstrate the models’ ability to
sufficient for Self-Descriptiveness
estimate overall testability from design
5)Availability of built-in test
information
function
Mustafa
3)The model is more practical in nature
6)Test restartability
3
2009
UML
having quantitative data on testability
7)Not sufficient for both structural
4)The software developer can use data to
and behavioural architecture
&
plan and monitor testing activities
Khan
5)The tester can use testability information
to determine on what module to focus
during testing
6)The software developer can use testability
metrics to review code, trying to find Main Issue J u l y 1 5 , 2 0 1 3 943 | | P a g e ISSN 22773061 944 | P a g e
J u l
refactoring that would improve the
testability of the code
1)tackle the regression test selection
1)Accountability 2)Accessibility
problem at the architecture/ design level in
3)Not sufficient for Self-
the context of UML-based development
Descriptiveness
al. 4. Conclusion This paper has aimed to provide an overview and compare recent progress in model testability. We study and survey several
approaches. But we cannot claim that these approaches are comprehensive and exhaustive. Finally, we have comparison
table with different columns that compares all the approaches. The main problem with most of these approaches to testing is
considering the behavioral architecture in model testability. It should be noted that although the above comparison is
conducted based on some prominent approaches, the outcome of this research is not restricted to such approaches. In other
words, my considered criteria either can be served as features to be included in a newly developing system or may be applied
to help generally evaluating or selecting model testability approaches. However the results of my comparison show that there
is no single superior model testability approach in all cases. Therefore, deciding which approach to use in a certain scenario
should be done based on its specifications and combine and present the new approach that is more comprehensive and
mature is my future work. 5. Acknowledgement This project is sponsored by Ministry of Higher Education (MOHE) in corporation with Universiti Teknologi Malaysia, Research
Management Centre (UTM-RMC) under Vote No: 03H74. The authors also would like to thanks those who are directly or
indirectly involved in this project. Main Issue 2)Higher efficiency in test selection based on 4)Not sufficient for both structural
the automation of design change analysis
and behavioural architecture
et
2009
UML
and traceability between UML designs and
4
Briand
regression test cases
3)Better support for assessing and planning
regression test effort earlier in the change
process that is once design changes have
been determined
1)synthesize a system state model of an
1)Accountability 2)Accessibility
object-oriented system from the relevant
3)Communicativeness 4)Not
& Mall
UML models
sufficient for Self-Descriptiveness
5
2)The synthesized state model is used to
5)Not sufficient for both structural
2009
UML
generate test specifications for transition
and behavioural architecture
Sarma
coverage
3)The model is used in a complementary
manner with traditional test techniques
4)The test specification generated by model
could detect bugs
1)Software contracts reduce the testing
1)Accountability 2)Accessibility
effort
3)Communicativeness 4)Not
2)Software contracts improve the testability sufficient for Self-Descriptiveness
of an object oriented class
5)Not sufficient for both structural
6
& Saha
3)Software contracts reduce the number of and behavioural architecture
test cases by 50% to test a class
2010
UML &
4)Software practitioners can make use of
Singh
Software
software contracts to reduce the testing
contract
effort
5)Software practitioners can make use of
software contracts to improve the testability
of the software
1)requirements model, design model, and
1)Accountability 2)Accessibility J u l y 1 5 , 2 0 1 3 944 | | P a g e ISSN 22773061 implementation quality model
3)Communicativeness 4)Not
(programming)
sufficient for Self-Descriptiveness
Rojas
2)notice process maturity and reusability
5)Retest efficiency
3)The model increase understanding of the 6)Not sufficient for both structural
&
2003
Quality model relationship between the attributes
and behavioural architecture
7
Ortega
(characteristics) and the subattributes
(subcharacteristics) of quality
4)the quality characteristics are: efficiency,
reliability, maintainability, portability,
usability and functionality
1)the usefulness of the documentation
1)Accountability 2)Accessibility
Deursen
the quality of the implementation
3)Not sufficient for Self-
2)the reusability and structure of the test
Descriptiveness
suite
4)Not sufficient for both structural
3)the suitability of the test tools used
and behavioural architecture
& Van
2006 Quality model the process capabilities
8
4)factors that influence the number of test
Bruntink
cases required testing the system
5)factors that influence the effort required
to develop each individual test case
6)test case generation factors
7)test case construction factors
& Bundell
1)Testability characteristics are: component 1)Accountability 2)Accessibility
traceability, component observability,
3)Communicativeness 4)Not
component controllability, component
sufficient for Self-Descriptiveness
9
2008
Test contracts
understandability, and component test
5)Not sufficient for both structural
Zheng
support capability
and behavioural architecture
2)improve model-based component
testability
Distinguish between internal quality,
1)Accountability 2)Accessibility
Jalbani
external quality and quality-in-use
3)Communicativeness 4)Not
sufficient for Self-Descriptiveness
10
2011
UML
5)Not sufficient for both structural
and behavioural architecture J u l y 1 5 , 2 0 1 3 945 | P a g e 945 | P a g e 945 | ISSN 22773061 Mohammadreza Najaftorkaman ,Pourya Nikfard, Maslin Masrom, Mohammadreza abbasy (2011), An efficient cryptographic Mohammad Reza Abbasy, Pourya Nikfard, Ali Ordi and Mohammad Reza Najaf Torkaman (2012), DNA Base Data Hiding
Algorithm, International Journal on New Computer Architectures and Their Applications (IJNCAA) 2(1): 183-192 The Society of
Digital Information and Wireless Communications, 2012 (ISSN: 2220-9085). 6. Reference Reza, H., Ogaard, K., & Malge, A. (2008). A Model Based Testing Technique to Test Web Applications Using Statecharts. Fifth International Conference on Information Technology: New Generations (itng 2008), 0-7695-309, 183–188. doi:10.1109/ITNG.2008.145 Puolitaival, O.-P., & Kanstrén, T. (2010). Towards flexible and efficient model-based testing, utilizing domain-specific
modelling. Proceedings
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1. doi:10.1145/2060329.2060349 Voigt, H., & Engels, G. (2008). Quality Plans for Measuring Testability of Models. Software Engineering (pp. 1–16). Retrieved
from www.vietnamesetestingboard.org Yeh, P., & Lin, J. (1998). Software Testability Measurements Derived from Data Flow Analysis Tatung Institute of Technology. 2nd Euromicro Conference on Software Maintenance and Reengineering (CSMR’98) (pp. 1–7). Kout, A., Toure, F., & Badri, M. (2011). An empirical analysis of a testability model for object-oriented programs. ACM
SIGSOFT Software Engineering Notes, 36(4), 1. doi:10.1145/1988997.1989020 Khalid, S., Zehra, S., & Arif, F. (2010). Analysis of object oriented complexity and testability using object oriented design
metrics. Proceedings
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1–8. doi:10.1145/1890810.1890814 Khan, R. a., & Mustafa, K. (2009). Metric based testability model for object oriented design (MTMOOD). ACM SIGSOFT
Software Engineering Notes, 34(2), 1. doi:10.1145/1507195.1507204 Briand, L. C., Labiche, Y., & He, S. (2009). Automating regression test selection based on UML designs. Information and
Software Technology, 51(1), 16–30. doi:10.1016/j.infsof.2008.09.010 Sarma, M., & Mall, R. (2009). Automatic generation of test specifications for coverage of system state transitions. Information
and Software Technology, 51(2), 418–432. doi:10.1016/j.infsof.2008.05.002 Pourya Nikfard, Mohammad Hossein Abolghasem Zadeh, Suhaimi Bin Ibrahim (2013), A Comparative Evaluation of
approaches for Web Application Testing, International Conference on Soft Computing and Software Engineering 2013
(SCSE'13), International Journal of Soft Computing and Software Engineering [JSCSE], ISSN: 2251-7545 & DOI:
10.7321/jscse, San Francisco, CA, USA. Pourya Nikfard, Harihodin Selamat, Mohd Naz‟ri Mahrin (2012), Functional Testing on Web Applications, Postgraduate Annual
Research on Informatics Seminar (PARIS 2012). Mohammad Reza Abbasy, Pourya Nikfard, Ali Ordi and Mohammad Reza Najaf Torkaman (2012), DNA Base Data Hiding
Algorithm, International Journal on New Computer Architectures and Their Applications (IJNCAA) 2(1): 183-192 The Society of
Digital Information and Wireless Communications, 2012 (ISSN: 2220-9085). Mohammad Reza Abbasy, Pourya Nikfard, Ali Ordi and Mohammad Reza Najaf Torkaman (2012), DNA Base Data Hiding
Algorithm, International Journal on New Computer Architectures and Their Applications (IJNCAA) 2(1): 183-192 The Society of
Digital Information and Wireless Communications, 2012 (ISSN: 2220-9085). 6. Reference Mohammadreza Najaftorkaman ,Pourya Nikfard, Maslin Masrom, Mohammadreza abbasy (2011), An efficient cryptographic J u l y 1 5 , 2 0 1 3 J u l y 1 5 , 2 0 1 3 | P a g e 946 | P a g e 946 ISSN 22773061 protocol based on DNA chip, © 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of GCSE
2011, Procedia Engineering. protocol based on DNA chip, © 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of GCSE
2011, Procedia Engineering. Mohammad Hossein Abolghasem Zadeh, Pourya Nikfard, Mohd Nazri Kama, Suhaimi Bin Ibrahim (2013), Software Changes:
Related Software Artifacts and their Relationships, The Second International Conference on Advances in Computing and
Networking (ACN - 2013), Bangkok, Thailand. Mohammad Hossein Abolghasem Zadeh, Pourya Nikfard, Mohd Nazri Kama, Suhaimi Bin Ibrahim (2013), Software Changes:
Related Software Artifacts and their Relationships, The Second International Conference on Advances in Computing and
Networking (ACN - 2013), Bangkok, Thailand. Mohammad Reza Najaf Torkaman, Pourya Nikfard, Nazanin Sadat Kazazi, Mohammad Reza Abbasy, and S. Farzaneh
Tabatabaiee (2011), Improving Hybrid Cryptosystems with DNA Steganography, E. Ariwa and E. El-Qawasmeh (Eds.): DEIS
2011, CCIS 194, pp. 42– 52, 2011. © Springer-Verlag Berlin Heidelberg 2011. Morteza Bagherpour, Pouria Nikfard, Arash Ghaed Mohammadi (2006), Hybrid Neural Network, Tabu search, Application to
single machine scheduling with earliness and tardiness penalty, Proceeding of the V international conference “System
Identification and Control Problems” SICPRO ‟06 Moscow‟ Morteza Bagherpour, Pouria Nikfard, Arash Ghaed Mohammadi (2006), Hybrid Neural Network, Tabu search, Application to
single machine scheduling with earliness and tardiness penalty, Proceeding of the V international conference “System
Identification and Control Problems” SICPRO ‟06 Moscow‟ Singh, Y., & Saha, A. (2010). Improving the testability of object oriented software through software contracts. ACM SIGSOFT
Software Engineering Notes, 35(1), 1. doi:10.1145/1668862.1668869 Singh, Y., & Saha, A. (2010). Improving the testability of object oriented software through software contracts. ACM SIGSOFT
Software Engineering Notes, 35(1), 1. doi:10.1145/1668862.1668869 Singh, Y., & Saha, A. (2010). Improving the testability of object oriented software through software contracts. ACM SIGSOFT
Software Engineering Notes, 35(1), 1. doi:10.1145/1668862.1668869 Ortega, M., & Rojas, T. (2003). Construction of a systemic quality model for evaluating a software product. Software Quality
Journal, 11:3(July), 219–242. Ortega, M., & Rojas, T. (2003). Construction of a systemic quality model for evaluating a software product. Software Quality
Journal, 11:3(July), 219–242. Bruntink, M., & Van Deursen, A. (2006). An empirical study into class testability. Journal of Systems and Software, 79(9),
1219–1232. doi:10.1016/j.jss.2006.02.036 Bruntink, M., & Van Deursen, A. (2006). An empirical study into class testability. Journal of Systems and Software, 79(9),
1219–1232. doi:10.1016/j.jss.2006.02.036 Zheng, W., & Bundell, G. (2008). ISSN 22773061 Contract-Based Software Component Testing with UML Models. Computer Science and its
Applications, 2008. CSA ’08. International Symposium on, 978-0-7695(13 - 15 October 2008), 83–102. Zheng, W., & Bundell, G. (2008). Contract-Based Software Component Testing with UML Models. Computer Science and its
Applications, 2008. CSA ’08. International Symposium on, 978-0-7695(13 - 15 October 2008), 83–102. Jalbani, A. A. (2011). Quality Assessment and Quality Improvement for UML Models. Jalbani, A. A. (2011). Quality Assessment and Quality Improvement for UML Models. Jalbani, A. A. (2011). Quality Assessment and Quality Improvement for UML Models. J u l y 1 5 , 2 0 1 3 947 | P a g e 947 | P a g e
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Primena koncepta "industrijski internet stvari" na primeru upravljivog električnog bojlera kao potrošača i analiza mogućnosti učešća u regulaciji učestanosti
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nikola.georgijevic@ekc-ltd.com чне речи — Регулација учестаности - Управљива потрошња - Интернет ствари - IIoT Ц2 06
ПРИМЕНА КОНЦЕПТА „ИНДУСТРИЈСКИ ИНТЕРНЕТ СТВАРИ“ НА ПРИМЕРУ
УПРАВЉИВОГ ЕЛЕКТРИЧНОГ БОЈЛЕРА КАО ПОТРОШАЧА И АНАЛИЗА
МОГУЋНОСТИ УЧЕШЋА У РЕГУЛАЦИЈИ УЧЕСТАНОСТИ
НИКОЛА ГЕОРГИЈЕВИЋ,
ЕЛЕКТРОЕНЕРГЕТСКИ КООРДИНАЦИОНИ ЦЕНТАР
ДУШАН ВЛАИСАВЉЕВИЋ,
ЕЛЕКТРОЕНЕРГЕТСКИ КООРДИНАЦИОНИ ЦЕНТАР
ВЛАДИМИР ШИЉКУТ,
ЈП ЕЛЕКТРОПРИВРЕДА СРБИЈЕ
ДЕЈАН МИСОВИЋ,
СИНКРОТЕК
САША МИЛИЋ,
ИНСТИТУТ НИКОЛА ТЕСЛА ДЕЈАН МИСОВИЋ,
СИНКРОТЕК
САША МИЛИЋ,
ИНСТИТУТ НИКОЛА ТЕСЛА 1
УВОД Циљ регулације учестаности и снаге размене у електроенергетском систему (ЕЕС) је
одржавање равнотеже између производње и потрошње електричне енергије. То се тренутно
постиже променом произведене снаге у циљу компензације неизбежних промена које су се
раније очекивале у потрошњи и врло ретко у производњи. Примарна регулација учестаности
представља спонтано тј. аутоматско дејство регулатора турбине. Секундарна регулација
представља накнадно суперпонирано дејство на примарну регулацију, преко улаза за тзв. спољну наредбу турбинских регулатора, ради елиминисања статичке грешке учестаности и/или
снага размене. У основи, секундарна регулација се реализује помоћу централног мрежног
регулатора регулационе области који одређује потребну производњу појединих регулационих
агрегата, на основу мерења учестаности и снага размене на интерконективним водовима [1]. Последњих година, сведоци смо релативно велике пенетрације обновљивих извора електричне
енергије (ОИЕ) у електроенергетски систем (ЕЕС) Србије. Иако је даљи раст удела ОИЕ у
производном миксу више него пожељан, они поседују извесне карактеристике, које са аспекта
регулације ЕЕС-а могу представљати извесне проблеме. Последњих година, сведоци смо релативно велике пенетрације обновљивих извора електричне
енергије (ОИЕ) у електроенергетски систем (ЕЕС) Србије. Иако је даљи раст удела ОИЕ у
производном миксу више него пожељан, они поседују извесне карактеристике, које са аспекта
регулације ЕЕС-а могу представљати извесне проблеме. р
у
ј
у р
р
На пример, производња ветроелектрана зависи од ветра. Стохастичка природа ветра може
изазвати нагле промене снаге ветроелектрана, што за последицу има варијације у снази размене
између ЕЕС Србије и осталих ЕЕС-â у оквиру ENTSO-E интерконекције. Подаци о
варијацијама снаге ветра и грешци снаге размене (АСЕ) на десетоминутним интервалима су
јавно доступни [2] и на основу тих података се могу лако уочити ситуације где се АСЕ мења по
градијенту већем од ±10 MW по минуту. Са друге стране, тренутна ситуација у ЕЕС Србије је
таква да секундарну регулацију учестаности и снаге размене готово искључиво врше јединице
које су у власништву ЈП ЕПС и то како хидроелектране, тако и термоелектране. Са циљем да се
прецизно одреде трошкови додатног хабања опреме у њима и цене коштања регулације
учестаности, гледано из угла произвођача, ЈП ЕПС је наручио студију [3] у којој су ови
трошкови процењени на основу искустава и процедура примењених на глобалном нивоу [4]–
[8]. Укратко, у [3] је закључено да учешће у регулацији учестаности може значајно да скрати
животни век опреме, нарочито код термо постројења. Приступ за решавање овог проблема је један од главних мотива за израду овог рада. 1
УВОД У раду је
приказан концепт за управљање електричним бојлерима, мада се концепт може проширити на
остале потрошаче који имају велике временске константе (црпне станице, системе за грејање и
хлађење…). Циљ је да се употребом управљиве потрошње смањи терет регулације учестаности
који је сада на произвођачу и пребаци на низ малих потрошача, те постигну одређени
економски бенефити за обе стране. После уводног поглавља, најпре је дат кратак приказ IIоT концепта и прототипа електричног
бојлера који је искоришћен за одређене експерименте (поглавље 3). На основу
експерименталних резултата, извршена је статистичка (бихевиорална) анализа употребе једног
електричног бојлера, а након тога, предложен је динамички модел већег броја потрошача и
извршене су симулације рада у секундарној регулацији учестаности. Економски ефекти
примене овог концепта приказани су у поглављу 8, док су закључне напомене дате у поглављу
9. СРБИЈА Кратак садржај — У овом раду приказан је прототип даљински управљивог прекидача
електричног бојлера са могућношћу очитавања вредности са сензора за мерење
температуре, струје и учестаности мреже. Полазећи од организације индустријског интернета ствари (Industrial Internet of Things -
IIOT), позиција управљивог бојлера, као потрошача, препозната је на ивичном нивоу. Системи SCADA, који служе за контролу и аквизицију управљиве потрошње и регулације
учестаности представљају следећи ниво управљања „магла―. Апликације задужене за
прорачуне и анализе података се налазе на апликативним серверима у нивоу облака. Након приказа наведеног прототипа, у раду је извршена статистичка анализа рада
електричног бојлера и дате су процене расположивих промена његове снаге и енергије -
повећања и смањења, зависно од тренутних потреба електроенергетског система. На
основу добијених статистичких показатеља, предложен је динамички модел великог броја
бојлера који се користе на сличан начин. Такав (збирни) модел је употребљен за анализу
учешћа већег броја електричних бојлера у регулацији учестаности. чне речи — Регулација учестаности - Управљива потрошња - Интернет ствари - IIoT 2
ИНДУСТРИЈСКИ ИНТЕРНЕТ СТВАРИ – IIoT Интернет ствари (Internet of Things – IoT) је спој виртуелног и физичког простора оријентисан
на потрошњу са циљем подизања квалитета услуга у скоро свим областима људске делатности. Нагли развој рачунарских и комуникационих система резултује великим протоцима и малим
временима кашњења информација, па је природно да се појаве IоT уређаји и системи који
унапређују постојеће и отварају широке области нових услуга. Уређаји IоT су често мобилни,
генеришу осредњу количину података без дефинисаног временског циклуса и непредвидиве
дужине трајања са релативно великим временским кашњењима. Индустријски Интернет ствари (Industrial Internet of Things – IIoT) представља надградњу IoT у
индустријском сектору. Основна особина IIоT је потпуна интеграција оперативних и 2 информационих технологија у индустријском окружењу где се генеришу велике количине
података уз строго дефинисане захтеве за поузданим преносом и малим кашњењем. Полазећи од предности ивичног рачунања (edge computing) које се извршава најближе изворима
података, што резултује најмањим кашњењима, у овом раду је предложена хибридна
концепција управљања потрошњом где се на ивичном нивоу, поред електричних бојлера налазе
и мале процесорске јединице за обраду података. Резултати обраде података ових јединица се
директно прослеђују на сервер у облаку чиме се практично реализује раванска концепција
управљања карактеристична за концепте IоT обраде података и управљања [9]. Имајући у виду
сложеност дистрибутивног система, у коме се налазе електрични бојлери као топлотно
акумулационе јединице, аутори рада предлажу хибридну IIoT концепцију (Сл. 1). у
р
ф
у
р
Полазећи од предности ивичног рачунања (edge computing) које се извршава најближе изворима
података, што резултује најмањим кашњењима, у овом раду је предложена хибридна
концепција управљања потрошњом где се на ивичном нивоу, поред електричних бојлера налазе
и мале процесорске јединице за обраду података. Резултати обраде података ових јединица се
директно прослеђују на сервер у облаку чиме се практично реализује раванска концепција
управљања карактеристична за концепте IоT обраде података и управљања [9]. Имајући у виду
сложеност дистрибутивног система, у коме се налазе електрични бојлери као топлотно
акумулационе јединице, аутори рада предлажу хибридну IIoT концепцију (Сл. 1). Рачунарскп-кпмуникаципна мрежа
Паметни
сензпри
Вишепараметарски
мпнитпринг
системи
Сервери
Панели за
визуелизацију
Алармне
јединице
Дистрибутивна ппрема
НИВО
ОБЛАКА
НИВО
МАГЛЕ
ИВИЧНИ
НИВО
ОБЛАК
Мрежа ивичних уређаја
Паметни
сензпри
Ивичне
јединице
за
управљаое
пптрпшопм:
Термпакумулаципни бпјлери
Ивични
сервер
ИВИЧНИ
НИВО
ВЕРТИКАЛНА ТРОСЛОЈНА КОНЦЕПЦИЈА
РАВАНСКА КОНЦЕПЦИЈА
Сл. 1 – IIoT хибридна концепција– спој вертикалне и раванске концепције
3
ПРОТОТИП
УРЕЂАЈА
ЗА
ДАЉИНСКО
ОЧИТАВАЊЕ
И
УПРАВЉАЊЕ НИВО
ОБЛАКА Алармне
јединице Панели за
визуелизацију НИВО
МАГЛЕ ИВИЧНИ
НИВО 3
ПРОТОТИП УРЕЂАЈА ЗА ДАЉИНСКО ОЧИТАВАЊЕ И УПРАВЉАЊЕ
ЕЛЕКТРИЧНИМ БОЈЛЕРОМ 3
ПРОТОТИП УРЕЂАЈА ЗА ДАЉИНСКО ОЧИТАВАЊЕ И УПРАВЉАЊЕ
ЕЛЕКТРИЧНИМ БОЈЛЕРОМ Прототип уређаја за даљинско очитавање и управљање електричним бојлером приказан је на
Сл. 2. Централни део прототипа је ESP32 микроконтролер (на доњој страни PCB-a) који садржи
два 32-битна Tensilica Xtensa LX6 језгра и модуле за Wi-Fi и Bluetooth комуникацију. ESP32
може поуздано да функционише у индустријским окружењима, са радном температуром у
распону од –40 °C до + 125 °C. Сл. 2 - Прототип IIоT уређаја за даљинско очитавање и управљање електричним бојлером Сл. 2 - Прототип IIоT уређаја за даљинско очитавање и управљање електричним бојлером Прототипом приказаним на Сл. 2 могуће је даљински очитавати температуру воде у бојлеру,
ефективну вредност струје, напона и учестаности електричне мреже на коју је овај прототип
прикључен. Поред функција за даљинско очитавање вредности са сензора, приказани прототип
садржи и релеј којим је могуће даљинско укључење, односно искључење електричног бојлера. Прототип је подешен тако да на секундном нивоу очитава мерења и преко Wi-Fi конекције
шаље резултате на сервер за даљу обраду. 4
СТАТИСТИЧКА АНАЛИЗА РАДА ЕЛЕКТРИЧНОГ БОЈЛЕРА 3 3 Прототип са Сл. 2 прикључен је на електрични бојлер капацитета 80 литара на коме су вршена
мерења у периоду од неколико месеци. Одлуком корисника, електрични бојлер ради у дневном
интермитентом режиму (рад бојлера је од 7 часова до 18 часова радним данима). Типичан
дневни дијаграм температуре воде и снаге електричног бојлера код кога су вршена мерења
приказан је на Сл. 3. Сл. 3 - Типичан дијаграм температуре воде и снаге електричног бојлера код кога су вршена мерења Сл. 3 - Типичан дијаграм температуре воде и снаге електричног бојлера код кога су вршена мерења Анализирањем Сл. 3, може се закључити да је референца температуре подешена на око 60°C. Занимљиво је да се ујутру бојлер укључује тек када температура воде падне значајно испод
60°C (то је усредњено на 7 часова ујутру), као и да је за загревање воде потребно релативно
доста времена. Заправо, време потребно за загревање воде може се врло добро проценити на
основу калориметријске једначине (уз занемаривање губитака кроз топлотну изолацију
бојлера): где је Q - количина топлоте изражена у џулима (J), m - маса воде у килограмима (kg), ΔT -
разлика температуре у целзијусима (°C), и c – специфични топлотни капацитет воде (4200
Ј/kg/K). 3
ПРОТОТИП УРЕЂАЈА ЗА ДАЉИНСКО ОЧИТАВАЊЕ И УПРАВЉАЊЕ
ЕЛЕКТРИЧНИМ БОЈЛЕРОМ Даље, полазећи од калориметријске једначине, a занемарујући губитке у топлотној изолацији и
познајући временски облик електричне снаге грејача, могуће је проценити количину топлоте
(ΔQo) у јединици времена (t) која се одводи из бојлера у различитим временским интервалима: ( )
( ) ( )
( ) Јединица мере за ΔQo/Δt је (J/s=W), тe је у овом раду ΔQo/Δt назначено са Po i названо снага
одвођења топлоте из електричног бојлера. Резултат естимације снаге одвођења приказан је на Сл. 4. Снага одвођења зависи од градијента
температуре, тј. висока је када температура воде у бојлеру нагло пада. Са друге стране,
температура расте када је грејач укључен, а Pel>Po. Сл. 4 - Приказ резултата естимације снаге одвођења топлоте (Po) у односу на температуру воде (Т) и ел. снагу (Pе) Сл. 4 - Приказ резултата естимације снаге одвођења топлоте (Po) у односу на температуру воде (Т) и ел. снагу (Pе) Коначно, на основу вредности Pо у времену, могуће је одредити статистичке показатеље у
облику кутијастих дијаграма приказаних на Сл. 5. На истој слици приказане су и расподеле Коначно, на основу вредности Pо у времену, могуће је одредити статистичке показатеље у
облику кутијастих дијаграма приказаних на Сл. 5. На истој слици приказане су и расподеле 4 температуре воде, као и средња вредност процентуалног времена укључености бојлера по
сатима. температуре воде, као и средња вредност процентуалног времена укључености бојлера по
сатима. Сл. 5 - Статистички показатељи одвођења топлоте, температуре воде и процента укључености Сл. 5 - Статистички показатељи одвођења топлоте, температуре воде и процента укључености На основу података са Сл. 5 можемо закључити да је нпр. у периоду око 11 часова очекивана
вредност потрошње топлоте око 1,0 kWh, док је очекивана вредност температуре воде око
55°C. Даље, ако се уведе претпоставка да је бојлер у неком дану у 11.00 ч. искључен, а да је
измерена температура воде 55°C, можемо закључити да тај бојлер, својим укључењем, може
обезбедити резерву електричне снаге за ЕЕС „на доле“, приближно у износу од 2 kW. Резерву
енергије у односу на очекивану потрошњу у том сату можемо проценити на основу мерене
температуре и промене референце температуре (Tref) коју може даљински да мења приказани
прототип (Сл. 1): ( ) Ако се референца температуре подеси на нпр. 80°C, резерва енергије износи око 2,33 kWh. 3
ПРОТОТИП УРЕЂАЈА ЗА ДАЉИНСКО ОЧИТАВАЊЕ И УПРАВЉАЊЕ
ЕЛЕКТРИЧНИМ БОЈЛЕРОМ Обрнуто, ако је бојлер укључен, променом референтне температуре на 45°C и својим
искључењем може обезбедити снагу резерве „на горе“ у износу 2 kW и енергију од 1,4 kWh у
односу на очекивану потрошњу. 5
ПРЕДЛОГ ДИНАМИЧКОГ МОДЕЛА ВЕЛИКОГ БРОЈА ЕЛЕКТРИЧНИХ
БОЈЛЕРА КОЈИ СЕ КОРИСТЕ НА СЛИЧАН НАЧИН 7 Пример управљања референцом температуре за случај од 500 бојлера. 5
ПРЕДЛОГ ДИНАМИЧКОГ МОДЕЛА ВЕЛИКОГ БРОЈА ЕЛЕКТРИЧНИХ
БОЈЛЕРА КОЈИ СЕ КОРИСТЕ НА СЛИЧАН НАЧИН Прва претпоставка је да је одређен број бојлера опремљен прототипом уређаја приказаним на
Сл. 2. Такви уређаји могу да обезбеде податке о тренутној температури воде у бојлеру, статус
укључености, а може и да му се даљински мења референца температуре. Друга претпоставка је да за све бојлере постоји статистичка расподела параметара по угледу на
Сл. 5. Пошто у овом раду располажемо мерењима са једног бојлера, претпоставиће се да се сви
електрични бојлери уклапају у статистичке показатеље са Сл. 5, тј. да се понашају слично, као и
да имају исту снагу грејача и исту запремину. ју
у
у р ј
у
р
у
Уводи се вектор променљивих стања Т, са димензијама једнаким броју бојлера: ̇
( ( ) ( )) где су и такође вектори, а статус укључености ( ( )) се мења у зависности од мерене
температуре воде и референце температуре сваког појединачног бојлера према функцији
хистерезиса приказаној на Сл. 6. Укратко, ако је грешка температуре већа од +5°C, бојлер се
укључује, ако је грешка мања од -5°C, бојлер се искључује. 5 5 Сл. 6 - Функција хистерезиса за одређивање статуса укључености бојлера
6
ПРЕДЛОГ УПРАВЉАЧКОГ АЛГОРИТМА Сл. 6 - Функција хистерезиса за одређивање статуса укључености бојлера
ПРЕДЛОГ УПРАВЉАЧКОГ АЛГОРИТМА Сл. 6 - Функција хистерезиса за одређивање статуса укључености бојлера Сл. 6 - Функција хистерезиса за одређивање статуса укључености бојлера
РЕДЛОГ УПРАВЉАЧКОГ АЛГОРИТМА у
ц ј
р
др ђ
6
ПРЕДЛОГ УПРАВЉАЧКОГ АЛГОРИТМА Управљачки алгоритам је замишљен за имплементацију на хипотетичком серверу у облаку који
прикупља све податке. Алгоритам најпре класификује бојлере на укључене и искључене, а
затим их сортира по доступној енергији „на горе“ и „на доле“. Приликом активације резерве
„на горе“, алгоритам искључује укључене бојлере са највишом температуром тако што шаље
нижу вредност референце температуре. Приликом активације резерве „на доле“, алгоритам
укључује искључене бојлере са најнижом температуром шаљући им вишу вредност референце
температуре. Пример оваквог управљања за ситуацију са 500 електричних бојлера дат је на Сл. 7. Ту су
приказани одзиви температуре на промену референце и процењене резерве снаге и енергије „на
горе“ и „на доле“. Када се свим бојлерима повећа вредност референце температуре, сви упале
грејач, те им снага резерве „на горе“ достигне максимум (500×2 kW=1 MW). Када температура
воде код већине достигне максималне вредности, енергија резерве „на доле“ приближи се нули. Обрнуто важи у случају обарања референце температуре. Сл. 7 Пример управљања референцом температуре за случај од 500 бојлера. Сл. 7
ДИНАМИЧКЕ СИМУЛАЦИЈЕ Динамичке симулације су извршене на четворомашинском систему који је доступан у [10]. Овај
систем се састоји од две потпуно симетричне области (А и Б), при чему је затечени ток снаге
413 MW из области А у област Б. На Сл. 8 је приказан резултат симулације испада 100 MW
производње у области Б. У први мах, грешка регулације износи око 50 MW, што је упола мање
од испале снаге. Разлог је што су области симетричне, те се једнако одазивају у процесу
примарне регулације учестаности – деле дебаланс од 100 MW. 6 6 Сл. 8 Симулација испада 100 MW у области Б, без управљиве потрошње Сл. 8 Симулација испада 100 MW у области Б, без управљиве потрошње Регулатор секундарне регулације је у овом моделу подешен тако да за око 90 секунди врати
снагу размене на вредност пре поремећаја. Ово је прилично брз одзив у односу на ситуацију
која се може затећи у пракси [2]. Регулатор секундарне регулације је у овом моделу подешен тако да за око 90 секунди врати
снагу размене на вредност пре поремећаја. Ово је прилично брз одзив у односу на ситуацију
која се може затећи у пракси [2]. Поред ове анализе, извршена је и анализа истог испада са претпоставком да је 100 хиљада
бојлера укључено у регулацију учестаности. Поређења ради, према попису из 2011. године,
само у Београду постоји око 600 хиљада домаћинстава. На Сл. 9 приказани су снага размене и
учестаност, а на Сл. 10 референце, температуре и доступна резерва управљиве потрошње. За
овај случај, снага размене се на референцу врати за око 20 секунди, што је значајно брже него у
случају без управљиве потрошње. Закључује се да је управљива потрошња итекако способна за
учешће у секундарној регулацији учестаности, при чему карактеристике овакве регулације могу
бити и боље него код конвенционалних јединица. Уочава се и да температуре воде
појединачних бојлера у овом периоду остају практично непромењене, те да је акумулисана
енергија практично непромењена у овом периоду. р
ј
р
р
у
р
ду
Сл. 9 Симулација испада 100 MW у области Б, са управљивом потрошњом
Сл. 10 Управљања референцом температуре над 100 хиљада бојлера у секундарној регулацији учестаности
8
ЕКОНОМСКИ ЕФЕКТИ Сл. 9 Симулација испада 100 MW у области Б, са управљивом потрошњом Сл. 9 Симулација испада 100 MW у области Б, са управљивом потрошњом
Сл. 10 Управљања референцом температуре над 100 хиљада бојлера у секундарној регулацији учестаности Сл. 9
ЗАКЉУЧАК У овом раду је предложен IIоT концепт управљиве потрошње заснован на управљању
електричним бојлерима. Приказан је прототип даљинског регулатора температуре воде бојлера,
као и резултати мерења на једном електричном бојлеру. Извршена је статистичка
(бихевиорална анализа) електричног бојлера као потрошача и процењене су статистичке
расподеле снаге потрошње воде по сатима. На основу ових података формиран је агрегирани
модел већег броја потрошача који се понашају на сличан начин и испитане су могућности
оваквог скупа потрошача за рад у секундарној регулацији учестаности. Показано је да је
оваквим системом могуће постићи чак и боље резултате у погледу времена одзива, те
растеретити постојеће, конвенционалне јединице које су укључене у секундарну регулацију. Поред техничких анализа, извршена је и упрошћена економска анализа једног оваквог пројекта. Истакнуто је да би се оваква инвестиција вратила за око 5 месеци, те да је стопа годишњег
прихода већ у првој години око 270%. 7
ДИНАМИЧКЕ СИМУЛАЦИЈЕ 9 Симулација испада 100 MW у области Б, са управљивом потрошњом Сл. 9 Симулација испада 100 MW у области Б, са управљивом потрошњом Сл. 10 Управљања референцом температуре над 100 хиљада бојлера у секундарној регулацији учестаности 8
ЕКОНОМСКИ ЕФЕКТИ 8
ЕКОНОМСКИ ЕФЕКТИ 7 7 Економски ефекти инсталације прототипа у постојеће електричне бојлере процењени су на
основу тренутне цене секундарне резерве, броја сати у години и инсталисане снаге електричног
бојлера. На основу збирне суме компоненти и цене израде плочице, процењена вредност израде
прототипа је око 2500 РСД по комаду за количине веће од 10.000 комада. Са ценом уградње,
која је врло једноставна, процењеном на 2500 РСД, укупна инвестиција по једном електричном
бојлеру износи око 5000 РСД. Са друге стране, само цена резерве снаге „на горе“ (без енергије) износи око 1525 РСД/MW/h,
тј. око 1,5 РСД/kW/h. Ако уведемо претпоставку да један бојлер снаге 2 kW може да пружи
1 kW резерве у сваком сату у години, долазимо до цифре од 13.359 РСД годишње на име
пружања резерве снаге. На основу овог упрошћеног прорачуна, закључује се да је повраћај ове
инвестиције око 5 месеци, тј. да је стопа годишњег прихода око 270%. Важно је истаћи да је предуслов за имплементацију оваквог система постојање правно-
регулативног оквира који омогућава улогу агрегатора потрошње као учесника на тржишту
електричне енергије који може пружати системске услуге, кроз објекат виртуелне електране. Виртуелна електрана представља агрегацију више дистрибуираних произвођача и/или
потрошача електричне енергије у један ентитет, посматрано из угла оператора система,
централно управљан и повезан у инфраструктури интелигентне мреже. Треба истаћи да
спроведена економска анализа уважава само директне трошкове укључења већег броја
електричних бојлера у један постојећи портфељ агрегатора, без уважавања индиректних
трошкова на рачун успостављања централног система виртуелне електране и осталих
придружених трошкова у вези са пословним процесом агрегатора. Key words — Load Frequency Control (LFC), Automatic Generation Control (AGC)
– Demand Response Management (DRM) – Industrial Internet of Things - IIoT. INDUSTRIAL INTERNET OF THINGS CONCEPT FOR DISTRIBUTED LOAD CONTROL
IMPLEMENTED ON ELECTRICAL WATER HEATERS WITH FREQUENCY CONTROL
ANALYSIS VLADIMIR ŠILJKUT,
JP EPS
DEJAN MISOVIĆ,
SINKROTEK
SAŠA MILIĆ,
INSTITUT NIKOLA TESLA
BEOGRAD VLADIMIR ŠILJKUT,
JP EPS
DEJAN MISOVIĆ,
SINKROTEK
SAŠA MILIĆ,
INSTITUT NIKOLA TESLA
BEOGRAD ЛИТЕРАТУРА [1]
Г. Радовић и други., “Примарна, секундарна и терцијарна регулација учестаности и
примарна регулација напона у ЕПС-у,” Зборник радова, Електротехнички институт
"Никола Тесла", 2010, бр. 20, стр. 179-200 [1]
Г. Радовић и други., “Примарна, секундарна и терцијарна регулација учестаности и
примарна регулација напона у ЕПС-у,” Зборник радова, Електротехнички институт
"Никола Тесла", 2010, бр. 20, стр. 179-200 ,
, р
,
р
[2]
Веб платформа “Energy Flux.” https://ems.energyflux.rs/#/dashboard (датум приступа
20.06.2021). [3]
Студија: “Aнализа и унапређење медологија за адекватно вредновање системских услуга
са прегледом методологија примењених у Jугоисточној Eвропи”, наручилац: ЈП ЕПС,
обрађивач: Електротехнички институт Никола Тесла, 2018. година. р ђ
р
у
[4]
P. Besuner and S. Lefton, “The cost of cycling coal fired power plants,” Coal Power Mag., pp. 16–20, 2006. [5]
National Energy Technology Laboratory, “Impact of Load Following on Power Plant Cost and
Performance : Literature Review and Industry Interviews,” p. 49, 2012, doi: DOE/NETL-
2013/1592. [6]
O. Nilsson and D. Sjelvgren, “Hydro unit start-up costs and their impact on the sh 8 8 scheduling strategies of swedish power producers,” IEEE Trans. Power Syst., vol. 12, no. 1, pp. 38–44, 1997, doi: 10.1109/59.574921. scheduling strategies of swedish power producers,” IEEE Trans. Power Syst., vol. 12, no. 1, pp. 38–44, 1997, doi: 10.1109/59.574921. [7]
E. Cabrera, V. Espert, and F. Mart, Hydraulic Machinery and Cavitation, vol. 1. Springer,
1996. [8]
EPRI извештај „Impact of Cycling on the Operation and Maintenance Cost of Conventional
and Combined-Cycle Power Plants“ 2013 година. [9] [9]
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stvari“, Zbornik radova XX međunarodnog naučno-stručnog Simpozijum Infoteh-Jahorina
2021, 20 (2021), Jahorina, Republika Srpska, 17-19 mart 2021, oznaka rada O-1.2 (30). ISBN
978-1-7281-8228-5. https://infoteh.etf.ues.rs.ba/papers.php [10]
„Performance of Three PSS for Interarea Oscillations“, документ доступан на:
https://www.mathworks.com/help/physmod/sps/ug/performance-of-three-pss-for-interarea-
oscillations.html 9 9 Војводе Степе 412, 11000 Београд
nikola.georgijevic@ekc-ltd.com SERBIA Abstract— This paper presents remote control and data acquisition prototype device
which is specially designed for electrical water heaters. The prototype is equipped with
the temperature, current, voltage and frequency transducers, but also with a power
relay. Hence, it can not only measure but also control the on/off state of the device. Starting from a hierarchical organization of Internet of Things organization (IoT),
position of a smart electrical water heater is recognized on the basic edge level. SCADA
systems used for control and acquisition of corresponding parameters represent the next
control level (fog). Applications used for automatic calculation and data analysis are
located in the cloud. After description of the prototype device and experimental results, statistical analysis
is performed aiming to explore the power consumption of the heater and the available
up and down energy and power reserve. On the basis of the statistical analysis results,
aggregated model of multiple electrical heaters is presented which can be used for the
power system dynamic analysis. This model is later used for the simulation of the
regular and hybrid automatic generation (and load) control (AGC) and the beneficial
influence of the controllable distributed load is highlighted. Key words — Load Frequency Control (LFC), Automatic Generation Control (AGC)
– Demand Response Management (DRM) – Industrial Internet of Things - IIoT. 10 U ovaj prostor ne unositi tekst. Ovaj prostor se koristi za potrebe unošenja oznaka za rad- U ovaj prostor ne unositi tekst. Ovaj prostor se koristi za potrebe unošenja oznaka za rad- НИКОЛА ГЕОРГИЈЕВИЋ, ДУШАН ВЛАИСАВЉЕВИЋ
ЕЛЕКТРОЕНЕРГЕТСКИ КООРДИНАЦИОНИ ЦЕНТАР ДЕЈАН МИСОВИЋ, СИНКРОТЕК ИНСТИТУТ НИКОЛА ТЕСЛА СРБИЈА Кратак садржај — У овом раду приказан је прототип даљински управљивог прекидача
електричног бојлера са могућношћу даљинског очитавања вредности добијених са
сензора за мерење температуре, струје и учестаности напона напајања. Полазећи од хијерархијске организације индустријског интернета ствари (Industrial
Internet of Things - IIOT), позиција процесорски управљивог бојлера, као потрошача,
препозната је на основном ивичном нивоу. Системи SCADA, који служе за контролу и
аквизицију параметара управљиве потрошње и регулације учестаности представљају
следећи виши хијерархијски ниво управљања „магла―. Апликације задужене за
аутоматске прорачуне и анализе података и параметара регулације учестаности се налазе Кратак садржај — У овом раду приказан је прототип даљински управљивог прекидача
електричног бојлера са могућношћу даљинског очитавања вредности добијених са
сензора за мерење температуре, струје и учестаности напона напајања. сензора за мерење температуре, струје и учестаности напона напајања. Полазећи од хијерархијске организације индустријског интернета ствари (Industrial
Internet of Things - IIOT), позиција процесорски управљивог бојлера, као потрошача,
препозната је на основном ивичном нивоу. Системи SCADA, који служе за контролу и
аквизицију параметара управљиве потрошње и регулације учестаности представљају
следећи виши хијерархијски ниво управљања „магла―. Апликације задужене за
аутоматске прорачуне и анализе података и параметара регулације учестаности се налазе
на апликативним серверима у нивоу облака. р
р
у
у
Након приказа наведеног прототипа, у раду је извршена статистичка анализа рада
електричног бојлера и дате су процене расположивих промена његове снаге и енергије -
повећања и смањења, зависно од тренутних потреба електроенергетског система. На
основу добијених статистичких показатеља, предложен је динамички модел великог броја
електричних бојлера који се користе на исти или сличан начин. Такав (збирни)
динамички модел је касније употребљен за анализу могућности учешћа већег броја
електричних бојлера у регулацији учестаности. Кључне речи — Регулација учестаности - Управљива потрошња - Интернет ствари - IIoT 11 11
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https://openalex.org/W2804334832
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https://link.springer.com/content/pdf/10.1007/s11205-018-1931-2.pdf
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Dutch Liberation Festivals: A Vehicle to More Politically Active Young Citizens, or Merely the Same Selective Audience?
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Social indicators research
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* Manja Coopmans
M.Coopmans@uva.nl Soc Indic Res (2019) 142:617–643
https://doi.org/10.1007/s11205-018-1931-2 1
Department of Child Development and Education, University of Amsterdam, Postbus 15776, 1001
NG, Amsterdam, The Netherlands
2
Department of Sociology and ICS (Inter‑University Centre for Social Science Theory
and Methodology), Utrecht University, Utrecht, The Netherlands 1
Department of Child Development and Education, University of Amsterdam, Postbus 15776, 100
NG, Amsterdam, The Netherlands Keywords Political engagement · Commemoration · Dutch liberation festivals ·
Socialisation · Voting · Youth 2
Department of Sociology and ICS (Inter‑University Centre for Social Science Theory
and Methodology), Utrecht University, Utrecht, The Netherlands 1 Introduction Political participation is considered an important aspect of active citizenship and crucial
for an effective democracy (Putnam 2000). According to Verba et al. (1995), political par-
ticipation can be described as “voluntary activities by ordinary people directed towards
influencing directly or indirectly political outcomes at various levels of the political sys-
tem” (p. 38). Over the past few decades, an intense debate soared in both the United States
and Europe regarding a potential decline in levels of political participation amongst young
people—at least when looking at more traditional forms such as voting (Fieldhouse et al. 2007; Putnam 2000; Russo and Stattin 2017; Sloam 2014). The last parliamentary elec-
tions in the Netherlands in 2017, for instance, showed that despite a significant turnout
(81.9%), the percentage of young people who voted (i.e. those between 18 and 24 years
old) dropped from 77% in the 2012 parliamentary elections to 67% in 2017 (NOS 2017), a
decline of almost 125,000 young citizens. Amongst youngsters who (had) followed a voca-
tional educational track (as compared to those in college or university education), turnout
was even lower. This falling voter turnout amongst young people is worrisome, as it means an unequal
representation of young people’s voices in politics, thereby threatening the democratic
ideal of political equality, and potentially resulting in young people becoming even more
marginalised from electoral politics. Moreover, the act of (not) voting is itself habit-form-
ing (Coppock and Green 2016; Plutzer 2002), meaning that not voting at a young age
decreases chances of voting in future elections. A prominent example of an attempt to
counter the declining political participation amongst young citizens is the promoting of
youth’s involvement in civic (i.e. non-political) activities, for instance by organising extra-
curricular citizenship activities at school (Geboers et al. 2013), or by stimulating them
to join voluntary organisations (McFarland and Thomas 2006). Recent studies, however,
show that civic involvement is often highly segregated, with higher educated, politically
engaged citizens more likely to join in civic activities (Sloam 2014; Van Ingen and Van der
Meer 2016). The quality of citizenship education offered at school also varies substantially
(Geboers et al. 2013; Manning and Edwards 2014), providing young people with unequal
chances of developing into politically active citizens. Dutch Liberation Festivals: A Vehicle to More Politically
Active Young Citizens, or Merely the Same Selective
Audience? Manja Coopmans1,2 Accepted: 17 May 2018 / Published online: 24 May 2018
© The Author(s) 2018 Abstract This article follows up on claims made on the motivating role of national com-
memorations for young people’s political participation. Cross-sectional data from a Dutch
adolescent panel are utilised to focus on a commemoration activity popular amongst young
people in the Netherlands, and empirically test to what extent participation in Dutch Lib-
eration festivals amongst young adults (aged 19–20 years old) is associated with their
inclinations to vote. To examine whether the association is spurious, several factors previ-
ously identified as important determinants of young citizens’ broader civic engagement are
accounted for, including parental communication about civic issues, citizenship activities
offered at school, involvement in voluntary organisations, and various sociodemographic
characteristics. Although the relationship between Liberation festival attendance and vot-
ing intentions is partially explained by a more general civic socialisation process, as indi-
cated by, amongst others, the role of parental civic communication and voluntary work, the
results show that Dutch Liberation festivals are positively associated with young people’s
voting intentions. Moreover, individuals with different educational trajectories or socioeco-
nomic backgrounds have similar chances of attending the Liberation festivals, highlight-
ing the potential of Dutch Liberation festivals to promote political participation amongst
all young people equally. At the same time, Liberation festivals are less often attended by
youth identifying with a non-Dutch ethnic background, thereby risking reinforcing gaps in
political engagement between youth with and without a migration background. Keywords Political engagement · Commemoration · Dutch liberation festivals ·
Socialisation · Voting · Youth 1 3 3 M. Coopmans 618 1 Introduction p g
p
y
A potentially interesting alternative are Dutch Liberation festivals, organised annually
on 5 May to commemorate the end of the Second World War in 1945, and the enduring
freedom in the Netherlands since. Although Dutch Liberation festivals are most popular for
the musical performances of famous artists, they are also meant to raise awareness of core
democratic rights and values such as freedom, tolerance, and respect. Dutch Liberation fes-
tivals are but one example of the many activities that are annually organised around the
world to commemorate the Second World War (Liu et al. 2005; McCrone and McPherson
2009). During the past decade, the discussion on whether the national past and the asso-
ciated (institutionalised) rituals to commemorate this past—that is, national commemora-
tions—are able to promote (democratic) citizenship values and behaviours, and political
participation in particular, has resurfaced (Elgenius 2011; Haskins 2015; Liu and Hilton
2005). Building on Durkheim’s (1912–1995) assumption that rituals have the ability to
reinforce shared beliefs within a society, several reports—especially in the educational sec-
tor—have connected commemoration of a national past to political engagement amongst
young citizens (Cowan et al. 2014; Van Nieuwenhuyse and Wils 2012). Empirical research
on the relationship between young citizens’ commemorative and political participation is,
however, scarce. 1 1 3 Dutch Liberation Festivals: A Vehicle to More Politically Active… 619 The combination of popular culture and content gives informal commemorations such
as Dutch Liberation festivals the potential to promote more political awareness amongst
all youth equally. Whereas more formal commemorations have been argued to be mainly
attended by an elite audience (Fox 2014), this is different for more informal commemo-
rative activities. The Liberation festivals are free of charge and organised in 14 different
cities across the country, thereby creating opportunities to reach a large segment of the
population. Annually, around one million citizens visit one of the 14 festivals, of which
50% is under the age of 25 (De Regt and Van der Lippe 2017). At the same time, the fact
that the festivals are most known for their music and take place only once a year makes it
questionable to what extent they can impact people’s behaviours. 1 Introduction An alternative explana-
tion of positive associations between young people’s festival attendance and their political
participation is therefore that both commemorative and political participation are outcomes
of a more general civic socialisation process (including both non-political and political
socialisation), and we are, in fact, looking at a spurious association. Utilising data from a representative sample of Dutch young adults (aged 19–20 years
old) from the Children of Immigrants Longitudinal Survey in the Netherlands (CILSNL),
the present study contributes to the existing literature by examining the extent to which
their participation in Dutch Liberation festivals positively influences their inclination
to vote. Structural equation modelling is applied to test the relationship between young
adults’ festival attendance and voting intentions whilst simultaneously including a number
of factors that have traditionally been considered important determinants of young adults’
broader civic engagement (Amnå 2012; Verba et al. 1995), including parental communica-
tion about civic issues, citizenship activities offered at school, involvement in voluntary
organisations, and various sociodemographic characteristics. In doing so, this study is one
of the first to structurally test whether (informal) national commemorations have the poten-
tial to promote young adults’ political participation, or whether we are merely looking at
the same ‘selective’ audience. 2.1 National Commemorations and Young Adults’ Political Participation National commemorations are institutionalised rituals organised on designated dates on
which a nation commemorates a defining event in its history as a nation (Schwartz 2015). According to Renan (1882–1990), the commemoration of a national trauma imposes a
sense of duty on citizens by staging experiences that emphasise the importance of com-
mon efforts to avoid repeating history. As such, national war commemorations have been
argued to work as civic lessons through which people come to accept and internalise soci-
etal norms, values, and responsibilities (Haskins 2015). This usage of commemorations
as ‘lessons from the past’ is also visible in many European war commemorations, Dutch
Liberation festivals included, that use memories of the Second World War to focus on
democratic values such as freedom, equality, and justice (Liu et al. 2005). At every Dutch
Liberation festival, for instance, a ‘Square of Freedom’ is present, where non-governmen-
tal organisations share stories of the past, such as the Netherlands Veterans Institute, and
inform visitors of current issues relating to the fragility of freedom in the Netherlands and
abroad, such as Amnesty International, the Red Cross, and ProDemos. During the many
musical performances, attention is also paid to past and present wars, for instance by the 3 M. Coopmans 620 ‘Ambassadors of Freedom’, who travel across the country to perform at several Liberation
festivals. ‘Ambassadors of Freedom’, who travel across the country to perform at several Liberation
festivals. In addition to functioning as a ‘lesson from the past’, it has been argued that com-
memorative rituals remind citizens of why they belong together by producing a momentar-
ily shared experience amongst its participants, heightening what Durkheim (1912–1995)
referred to as ‘collective effervescence’. Supposedly, the temporary sense of collective
consciousness that follows from participating in commemorative rituals not only carries
over into more persistent feelings, but also increases an urge to act and contribute to the
group’s—or in this case society’s—well-being (see Collins 2004 for a review). Dutch Lib-
eration festivals try to accomplish a feeling of shared belonging during the ‘Five to Five
moment’, in which visitors of all 14 festivals are invited to dance simultaneously to the
same song and ‘pass along the freedom’, which is symbolised by large balloons that are
let loose over the audience. The ‘Fire of Liberation’ that is carried across the country and
burns at all festivals is another reference to a shared national past. 2.1 National Commemorations and Young Adults’ Political Participation The combination of a heightened sense of duty and increased awareness of democratic
values and the importance of a democratic system, which supposedly follow from attend-
ing a Dutch Liberation festival, increase one’s chances of translating the above described
urge to act, which is also supposed to follow from participating in commemorative ritu-
als, into concrete actions to contribute to the continuance of democracy, of which political
participation is considered a key aspect (Putnam 2000). This reasoning is consistent with
behavioural theories such as Ajzen’s (1991) theory of planned behaviour. Obviously, many
forms of political participation exist. One of the most visible forms that adolescents grow
up with in a democracy, however, is voting in local, national, and international elections. In the Netherlands, for instance, municipality and parliamentary elections are held every
4 years, and elections for the European parliament every 5 years. Voting is also one of the
least demanding forms of political participation compared to, for instance, working for a
political campaign or participating in a political protest (Verba et al. 1995), and therefore
a logical first step for young people in the process of political participation. We therefore
hypothesise that: Young people who have attended a Dutch Liberation festival are more
inclined to vote (H1). 2.2.1 The Home Environment: Parental Civic Communication 2.2.1 The Home Environment: Parental Civic Communication Traditional socialisation theories often consider the family—and the primary caregivers,
most often the parents, in particular—as the most likely agents of socialisation during ado-
lescence (Glass et al. 1986; Parsons and Bales 1956). Literature focusing more specifically
on civic participation has also highlighted the crucial role of parents’ civic orientations in
their children’s civic socialisation process (Beck and Jennings 1982; Verba and Nie 1972). Following up on this, numerous studies have shown that parents exert a substantial influ-
ence on the civic attitudes and behaviours of their children, especially during childhood
and adolescence (Hooghe and Boonen 2015; Jaspers et al. 2008). One way in which this
learning process may commence is through communication. Talking about civic issues or
events in everyday life not only teaches children about important societal topics, but also
clarifies their parents’ values and beliefs, which increases the likelihood of children adopt-
ing similar attitudes and behaviours (Jennings et al. 2009; Quintelier 2015b). g
g
Moreover, it creates an environment in which children are actively familiarised with a
civic engagement, including, as discussed, not only political attitudes and behaviours, but
also social and cultural ones. Schmid (2012), for instance, found that, in family environ-
ments in which discussions about civic issues were more frequent, adolescents were also
more aware of what it meant to act in a socially responsible way, a concept closely related
to one of the key aspects of civic engagement, namely the willingness to be committed
to the well-being of a larger group (Sherrod et al. 2002). Talking about civic topics with
one’s parents can thus be expected to heighten levels of civic engagement, thereby increas-
ing chances of participating in activities expressing this engagement, for instance through
voting, but also through participation in nationally organised commemorations. We there-
fore hypothesise that: The association between young people’s previous Liberation festival
attendance and their inclination to vote is partially explained by parental civic communi‑
cation (H2). 2.2 Alternative Explanations of Associations Between Commemorative
and Political Participation An alternative explanation of positive associations between young people’s Liberation fes-
tival attendance and their inclinations to vote is that the association is caused by other fac-
tors predicting both commemorative and political participation, that is, a spurious relation-
ship. In this study, we distinguish between two types of alternative explanations. First, we
are interested in the extent to which commemorative and political activities are outcomes
of a more general civic socialisation process. In this case, commemorative and political
participation are both considered expressions of a certain level of civic engagement, a term
used to describe a wide range of citizenship attitudes and behaviours, including, but not
limited to, participation in political, social, and even cultural activities (Adler and Gog-
gin 2005; Amnå 2012). We look at three environments that are thought to be crucial for
young people’s civic socialisation: the home, the school, and the out-of-school (i.e. leisure
time) environment. In all three cases, we focus on socialisation forms that directly address
civic issues: by communicating with one’s parents about social or political issues, via citi-
zenship education at school, or through involvement in voluntary organisations. Second, Dutch Liberation Festivals: A Vehicle to More Politically Active… 621 to examine selection processes as an alternative explanation of associations between com-
memorative and political participation, we consider several sociodemographic characteris-
tics that previous studies have shown to be particularly relevant in predicting levels of civic
engagement, including, amongst others, educational trajectory, socioeconomic status, and
ethnic origin (Verba et al. 1995). 2.2.2 The School Environment: Extra‑Curricular Citizenship Activities 2.2.2 The School Environment: Extra‑Curricular Citizenship Activities Another important socialising agent facilitating the civic socialisation process of young
citizens is the school. Not only does general education aid the development of basic knowl-
edge and skills necessary for civic engagement (Verba et al. 1995), citizenship has, since
the 1990s, been introduced as a compulsory school subject in almost all European coun-
tries, the Netherlands included (Eurydice 2012). One of the main goals of citizenship
education in the Netherlands is to foster active citizenship amongst young people, which
includes, amongst others, active political participation, but also being able to fulfil social,
everyday life tasks that are part of being a citizen, such as acting in a socially responsible
manner, and commitment to manifestations of Dutch culture (Ten Dam et al. 2011; Ten
Dam and Volman 2007). It is, however, left to schools to decide to what extent and in
what way they want to accomplish this. Hence, young people’s citizenship outcomes can be 1 3 3 3 M. Coopmans 622 expected to differ depending upon the emphasis that is placed upon citizenship education
by the school the student attends.f expected to differ depending upon the emphasis that is placed upon citizenship education
by the school the student attends.f Although empirical research on the effects of citizenship education on young people’s
civic engagement has provided mixed evidence, extra-curricular citizenship activities
(i.e. additional student activities supervised by the school to help foster active citizenship
amongst their students), such as voluntary service activities or a visit to parliament, seem
promising, at least where the political aspect of citizenship behaviour is concerned (Gebo-
ers et al. 2013). This form of ‘learning by doing’ offers students active, participative expe-
riences that help them acquire both a more realistic picture of what civic engagement looks
like, as well as an opportunity to practise specific skills that will help them develop into
civically active citizens. Supporting this line of argumentation, students participating in
extra-curricular citizenship activities such as a school council or a visit to parliament or a
museum have been found to be more interested in civic affairs and social problems, as well
as being more politically active later in life (Geboers et al. 2013; Hoskins et al. 2012; Keat-
ing and Janmaat 2016). 2.2.3 The Out‑of‑School Environment: Involvement in Voluntary Organisations 2.2.3 The Out‑of‑School Environment: Involvement in Voluntary Organisations Extra-curricular activities outside school (i.e. organised by other institutions) have also
been shown to promote political participation amongst adolescents, a common exam-
ple being involvement in voluntary organisations (Marzana et al. 2012; McFarland and
Thomas 2006; Quintelier 2008, 2015a). Voluntary organizations are argued to be power-
ful political socialisation agents engaging young people in politics through various mecha-
nisms. In addition to the advantages of extra-curricular activities for young people’s politi-
cal participation that were described in the previous section, voluntary organisations also
promote a sense of community and institutional trust which, according to theories on social
participation (Cassel 1999; Olsen 1972), motivate people to participate in other civic and
political activities (Cicognani et al. 2012). A third argument often used to underline the
political or civic value of voluntary organisations is that of relational support, referring to
the facilitating (or: recruiting) role of relationships with other members of the organisation
(McFarland and Thomas 2006; Sidney Verba et al. 1995). Based upon these arguments,
volunteering can be expected to show positive associations with both festival attendance
and voting intentions (many Liberation festivals are even partially run by volunteers). We
therefore hypothesize that: The association between young people’s festival attendance and
their inclination to vote is partially explained by their involvement in voluntary organisa‑
tions (H4). 2.2.2 The School Environment: Extra‑Curricular Citizenship Activities Following this line of argumentation, young people who attended a
school that paid more attention to citizenship education in the form of extra-curricular citi-
zenship activities can be expected to develop into more actively participating citizens, both
in terms of commemorative and political participation. Hence, we hypothesise that: The
association between young people’s previous Liberation festival attendance and their incli‑
nation to vote is partially explained by the extra-curricular citizenship activities offered at
school (H3). 2.2.4 Sociodemographic Factors In addition to the forms of civic socialisation discussed above, previous research on civic
participation has identified several sociodemographic characteristics that determine lev-
els of civic engagement in general and electoral participation in particular. Building on 1 3 623 Dutch Liberation Festivals: A Vehicle to More Politically Active… Bourdieu’s theory of social and cultural reproduction (Bourdieu 1984; Bourdieu and Pas-
seron 1977), studies have shown that young people from families with more resources
have higher chances of becoming politically active citizens (McFarland and Thomas 2006;
Sloam 2014). Two of the most consistent resources are socioeconomic status and level
of education (Bovens and Wille 2010; Verba et al. 1995). A higher educational level and
socioeconomic status have also been found to positively predict various forms of commem-
orative participation (Lubbers and Meuleman 2016). In addition to economic resources,
Bourdieu mentions the importance of cultural resources, of which highbrow activities such
as reading literature and visiting museums are prominent examples (De Graaf et al. 2000). Such activities can be expected to not only signal but also intensify one’s civic engage-
ment, including—as discussed before—political attitudes and behaviours, as well as an
interest in the national past and its commemorations (see, for instance, Cowan and Mai-
tles 2011; Savenije and De Bruijn 2017). Hence, we expect that: The association between
young people’s previous Liberation festival attendance and their inclination to vote is par‑
tially explained by their level of education (H5a), the socioeconomic status of their parents
(H5b), their reading activities (H5c), and their museum activities (H5d). g
Another demographic characteristic we believe to be relevant when examining rela-
tionships between commemorative and political participation is migration background. Previous studies have shown lower levels of political engagement amongst citizens with a
migration background, often explained by lower levels of socio-cultural integration, socio-
economic status, and feelings of identification with the politics of the country of origin (De
Rooij 2012; Sanders et al. 2014; White et al. 2008). Differences in voting behaviours are
less apparent for children of immigrants (Humphries et al. 2013; Quintelier 2009). Lev-
els of commemorative participation are also lower amongst citizens with a (non-Western)
migration background (Coopmans et al. 2016). This is not surprising: Second World War
commemorations in Europe are often focused on the history of the native population, as
large-scale immigration did not start until after the war (Messina 2007). 2.2.4 Sociodemographic Factors For young peo-
ple born in the Netherlands, the extent to which their migration background impacts their
participation can be expected to depend on their identification with their ethnic origin, as
well as their identification with the Netherlands (see also Coopmans et al. 2015). We there-
fore hypothesise that: The association between young people’s previous Liberation festival
attendance and their inclination to vote is partially explained by their ethnic (H6a) and
national identification (H6b). 3.1 Data We make use of the fifth wave of the ‘Children of Immigrants Longitudinal Survey in
the Netherlands’ (CILSNL), which is a continuation of the Dutch part of the ‘Children
of Immigrants Longitudinal Survey in Four European Countries’ (CILS4EU). The panel
study describes the life courses of adolescents of native and immigrant descent in the Neth-
erlands. The first wave was collected in 2010 amongst 14-year-old adolescents in their third
grade of secondary school. A three-stage stratified sample design was applied, with an
oversampling of schools with a high share of students of non-Western origin. The initial
response rate amongst schools in the Netherlands was 34.9%. To increase this participation
rate, schools that refused were replaced with schools highly similar to the initially sampled 3 M. Coopmans 624 schools, leading to a school-level participation rate of 91.7%, with a class-level participa-
tion rate of 94.5% and a student-level participation rate of 91.1%. In total, 4963 pupils in
252 classes in 118 schools participated in the first wave, including a subset of 600 stu-
dents who were not part of the original sampling frame (for more information on sampling
design and response rates, see CILS4EU 2016). As changes in class composition between
the third and fourth year of secondary school are common in the Netherlands, in the second
wave, schools were asked to participate with all fourth-grade classes that held initial first
wave respondents. Consequently, 2118 additional students were interviewed. In subsequent
waves, all 7081 respondents were approached. Information on our main variables of interest, commemorative and political participa-
tion, was collected in the fifth wave of CILSNL in 2015 (Jaspers and van Tubergen 2015).1
In this wave, all respondents were at least 18 years old, and thus allowed to vote. In total,
3836 respondents participated in wave 5. To overcome power issues and ensure a large
enough sample size, we initially kept all (trustworthy) respondents who completed the
full version of the questionnaire, which included items on commemorative and political
participation (N = 3761) .2 Information on citizenship activities offered at the participat-
ing schools during the time respondents were still in school (i.e. wave 1–2) was collected
in 2016 in the CILSNL Citizenship Education project (Coopmans 2016). To merge the
school-data with the individual-level data, information from the earlier waves was used. Information on citizenship activities organised at school was available for 1935 respond-
ents. 3.1 Data Respondents with missing values on our variables of interest were excluded. Most
missing values were found for parental civic communication (N = 235) and parental socio-
economic status (N = 224). In total, 1512 respondents had valid information on all variables
of interest, originally sampled from 58 different schools. Although a drastically smaller
sample size than our initial sample, this is the first dataset that allows for the testing of
associations between young adults’ commemorative and political participation, whilst con-
sidering potentially confounding factors at the individual and school level. 1 Since this topic was only covered in wave 5, we were unable to analyse changes in commemorative and
political participation. The panel structure was, however, valuable in that it allowed us to examine relation-
ships between previous socialising environments (i.e. in wave 1–2) and civic engagement several years later
(i.e. wave 5). For the construction of educational track, parental socioeconomic status, and parental civic
communication, data from the first four waves was used as well (Jaspers and van Tubergen 2014; Kalter
et al. 2016a, b, c). For a more extensive overview of which wave was used for which measurement, see
Table 3 in the “Appendix”.
2 In total, 72 respondents completed a shortened version of the questionnaire. Moreover, three respondents
were excluded because of untrustworthy answers. n total, 72 respondents completed a shortened version of the questionnaire. Moreover, three respondents
e excluded because of untrustworthy answers. Table 3 in the “Appendix”. 3.2 Measures Voting intentions our dependent variable, was measured by asking respondents in wave 5:
“If parliamentary elections were held today, for which party would you vote?” Answer cat-
egories comprised all major parties in the Netherlands, as well as the options ‘other party’,
‘I don’t know’, ‘blank’, and ‘I would not vote’. A dichotomous variable was created, distin-
guishing between people who indicated that they would not vote, and people who intended
to vote, including those voting ‘blank’. Since we do not know whether respondents who
responded with ‘I don’t know’ were unsure of whether they would vote or of which party 1 3 Dutch Liberation Festivals: A Vehicle to More Politically Active… 625 they would vote for (i.e. if they would fall in the category voters or non-voters), they were
coded as missing and excluded from our analyses (N =364).3 they would vote for (i.e. if they would fall in the category voters or non-voters), they were
coded as missing and excluded from our analyses (N =364).3 Participation in Dutch Liberation festivals was measured by asking respondents in wave
5 how often in the past years they had visited a Liberation festival on 5 May. Response cat-
egories were: (0) ‘never’; (1) ‘sometimes’; (2) ‘almost always’. As we are more interested
in factors predicting whether young adults attend at all than we are in factors that predict
whether adolescents attend every year versus only once or twice, a dichotomous variable
was created, distinguishing between respondents who had never attended a Liberation fes-
tival versus respondents who had either sometimes or almost always attended a Liberation
festival. Parental civic communication was measured using information from wave 2, thereby
providing an indication of the civic home environment when respondents, who at the
time of the interview were between 16 and 17 years old, still lived at home. Respondents
were asked: “How often do your parents talk with you about political and social topics?”
Response categories were: (0) ‘every day’; (1) ‘at least once a week’; (2) ‘at least once a
month’; (3) ‘less often’; (4) ‘never’. Response categories were recoded so that higher val-
ues indicated more frequent communication.f Extra-curricular citizenship activities offered at school were measured at the school-
level. In an additional data collection, schools were asked whether they had organised the
following extra-curricular citizenship activities in the school year 2010/2011 (the year
in which the first data were collected) for the students who participated in the CILSNL
data collection, i.e. wave 1): (a) elections; (b) debates; (c) visit to parliament; (d) other
democracy related excursions or museum visits; (e) a democracy related guest lecture;
(f) an extra exam course on social sciences; (g) student council. A distinction was made
between activities that took place outside the school environment (i.e. visits to parliament
and other excursions) and activities that took place inside the school environment (i.e. elec-
tions, debates, and guest lectures). The extra social sciences course and student council
were included as separate items. 3 Additional (multinomial logistic) analyses in which we did include respondents who did not yet know
what to vote resulted in similar findings. 1 3 Together, these four variables provided an indication of
the civic school environment when respondents still attended (secondary) school. Voluntary work was measured by asking respondents in wave 5: “In your spare time,
how often do you do voluntary or community work?”. Answer categories were: (0) ‘every
day’; (1) ‘at least once a week’; (2) ‘at least once a month’; (3) ‘less often’; (4) ‘never’. Response categories were recoded so that higher values indicated more frequent volun-
teering. A categorical variable was constructed, distinguishing between those volunteering
structurally (i.e. at least once a month), those volunteering occasionally (i.e. less often than
once a month), and those volunteering never. The latter acted as the reference category. Educational trajectory was operationalised as the last known level of education, meas-
ured with the question “What is, at this moment, your most important activity?” Response
categories were: (a) ‘secondary school’; (b) ‘intermediate vocational education’; (c)
‘higher vocational or college education’; (d) ‘university’; (e) ‘working’; (f) ‘unemployed’;
(g) ‘something else’. For those respondents answering (e), (f), or (g), we used information
from earlier waves to construct their latest known educational trajectory (see Appendix
Table 3). We distinguished between three tracks: (0) vocational (preparatory/intermediate
vocational education); (1) college (higher general and college education); (2) university 1 3 M. Coopmans 626 (university preparatory education and university). Those in the vocational track acted as
the reference category. (university preparatory education and university). Those in the vocational track acted as
the reference category. Parental socioeconomic status was taken into account using the International Socio-
Economic Index of Occupational Status (ISEI) score of the mother or father of the respond-
ent (Ganzeboom et al. 1992) as reported by the parents themselves in wave 1 and 2. The
highest score applied. If parental information was missing, parental occupation as reported
by their children (i.e. the respondents) in wave 1 and 2 was used. Reading activities was measured by asking respondents in wave 5 how often they read a
book (not for school), and museum activities by asking how often respondents went to the
museum. Answer categories for both questions were: (0) ‘every day’; (1) ‘at least once a
week’; (2) ‘at least once a month’; (3) ‘less often’; (4) ‘never’. Response categories were
recoded so that higher values indicated more frequent reading or museum activities. 1 3 Simi-
lar to our volunteering variable, two categorical variables were constructed, both distin-
guishing between structural, occasional, and never.i Ethnic identification was included by asking respondents in wave 5 to which non-Dutch
group(s) they felt they belonged. Answer categories were: (0) ‘no other group’; (1) ‘Turk-
ish’; (2) ‘Kurdish’; (3) ‘Moroccan’; (4) ‘Berbers’; (5) ‘Surinamese’; (6) ‘Hindu’; (7) ‘Cre-
ole’; (8) ‘Javan’; (9) ‘Chinese’; (10) ‘Curacao’; (11) ‘Aruban’; (12) ‘Antillean’; (13) ‘Indo-
nesian’; (14) ‘other group’. A dummy variable was included distinguishing between those
who did not identify with a non-Dutch group and those who did.i National identification was included by asking respondents in wave 5: “How strongly do
you feel Dutch?”. Answer categories were: (0) ‘very strongly’; (1) ‘fairly strongly’; (2) not
very strongly; (3) not at all strongly’. Response categories were recoded so that higher val-
ues indicated higher levels of national identification. Due to the small number of respond-
ents responding with ‘not at all strongly’, a categorical variable was constructed in which
those identifying ‘not very strongly’ and ‘not at all strongly’ were combined in one group
that acted as the reference category. Controls To control for other sociodemographic factors, information was included on
respondents’ gender, whether respondents still lived with their parents, and the degree of
urbanisation of the school location. Gender was operationalized as a dummy variable for
male, living with parents as a dummy variable for respondents still living with their parents
in wave 5, and urbanisation as the size of the municipality where the school was located
that respondents attended in wave 1 (i.e. a continuous, school-level variable, indicating the
number of citizens/10,000). 4.1 Descriptive Results Table 1 depicts descriptive information of our sample of 1148 young adults, the majority
between 19 and 20 years old. The findings show that 87% intended to vote in the next elec-
tions, whilst 13% indicated not to want to vote. Taking the young adults who indicated to
not yet know what political party they would vote for into account (N = 364), the statistics
are comparable to the percentage of young people (i.e. below 24 years old) who voted in
the last parliamentary elections in the Netherlands, namely 67% (NOS 2017). About 60%
of our sample had visited a Dutch Liberation festival at least once over the past years. On average, respondents had talked to their parents about political and social issues at
least once a month. A little under half of the respondents was involved in voluntary organi-
sations, either occasionally or structurally. Of the extra-curricular citizenship activities that
could be offered at school during the school year 2010/2011, schools most often organized
either a visit to parliament or other type of excursion (87%), a student council (79%), or an
activity inside the school environment, such as a debate, mock elections, or a guest lecture
(76%). Around half of the schools offered an additional exam course in social sciences.i f
An examination of educational track indicated 43% had finished or was currently in the
(preparatory) vocational track, whilst 57% was in the (preparatory) college or university
track. The former group is therefore somewhat under-represented compared to the general
Dutch population between 15 and 25 years old with at least primary education (Statistics
Netherlands 2016). Respondents’ parents had, on average, an ISEI score of 44, which can
be considered a medium socioeconomic status. Moreover, the majority of respondents read
books in their leisure time (70%), and a little over half of the respondents had visited a
museum (57%). Finally, 20% of the respondents identified with a non-Dutch ethnic group,
and most identified fairly to very strongly with Dutch society. 3.3 Analytical Strategy Since we wanted to test a structural model in which festival attendance was both an inde-
pendent variable predicting voting intentions, and a dependent variable predicted by civic
socialisation measures and sociodemographic variables, generalised structural equation
modelling was applied using Stata, version 15 (StataCorp 2017). Moreover, given that the
data had a hierarchical structure (i.e. respondents within schools) and the models included
not only individual characteristics but also school characteristics (i.e. citizenship activi-
ties offered at school), multilevel models were analysed. Finally, considering the dependent
variables were dichotomous, logistic models were examined. Intercept-only models found
an intraclass correlation of .129 for voting and .180 for festival attendance, meaning that
13 and 18% of the variation in voting intentions and festival attendance respectively can
be attributed to the school that adolescents attended. As unobserved heterogeneity affects Dutch Liberation Festivals: A Vehicle to More Politically Active… 627 coefficients differently in logistic regressions than in linear regressions, it is difficult to
interpret (log) odds ratios as substantive effects, or to compare them across models with
different independent variables (Mood 2010). We therefore calculated and reported average
marginal effects (AMEs), which are not only more easily interpretable in terms of sub-
stantive effect sizes than odds ratios, but also unaffected by unobserved heterogeneity that
is unrelated to the independent variables in the models and therefore comparable across
models. AMEs indicate the change in the probability of a respondent voting in the next
elections (versus not voting) or having attended a Liberation festival (versus having never
attended a Liberation festival), for every one-unit change in an explanatory variable, esti-
mated over all the possible values of the variables in the model. 4.2 Explanatory Results Table 2 shows the AMEs of the multilevel logistic structural equation models estimat-
ing young adults’ voting intentions and previous attendance at Dutch Liberation festivals. In Model 1, in which we included previous festival attendance as a potential predictor of
future voting intentions (whilst controlling for gender, living with parents, and degree of
urbanisation), we found a significant positive effect: Young adults who had visited a Lib-
eration festival over the past years had a 7% higher chance of voting in the next elections 3 3 M. Coopmans 628 Table 1 Descriptive statistics of
the variables
Variables
Min. Max. Mean
SD
Individual-level (N = 1148)
Voting intentions
0
1
.87
Liberation festival attendance
0
1
.59
Parental civic communication
0
4
2.17
1.23
Voluntary work
Never
0
1
.54
Occasionally
0
1
.30
Structurally
0
1
.16
Educational track
Vocational
0
1
.43
College
0
1
.30
University
0
1
.27
Parental ISEI score
0
88.96
43.97
19.94
Reading activities
Never
0
1
.30
Occasionally
0
1
.36
Structurally
0
1
.34
Museum activities
Never
0
1
.43
Occasionally
0
1
.50
Structurally
0
1
.07
Ethnic group identification
0
1
.20
National identification
Not very strongly
0
1
.10
Fairly strongly
0
1
.40
Very strongly
0
1
.50
Gender (male)
0
1
.42
Living with parents
0
1
.74
School-level (N = 57)
Citizenship activities in school
0
1
.76
Citizenship activities outside school
0
1
.87
Extra social sciences course
0
1
.54
Student council
0
1
.79
Urbanisation
1.18
77.36
14.70
2.18 Table 1 Descriptive statistics of
the variables Table 1 Descriptive statistics of
the variables (versus not voting) compared to young adults who had never visited a festival, which is
consistent with Hypothesis 1. (versus not voting) compared to young adults who had never visited a festival, which is
consistent with Hypothesis 1. In Model 2, 3, and 4, we included three alternative explanations of associations between
young adults’ previous festival attendance and future voting intentions, all indicators of a
more general civic socialisation process: the civic environment at home, measured by the
frequency of civic communication with one’s parents (Model 2), the civic environment at
school, indicated by the extra-curricular citizenship activities offered (Model 3), and the
civic environment out-of-school, measured by their involvement in voluntary organisations
(Model 4). 4.2 Explanatory Results The inclusion of parental civic communication slightly decreased the AME of 1 3 1 3 Dutch Liberation Festivals: A Vehicle to More Politically Active… 629 e 2 Multilevel logistic generalised structural equation model predicting voting intentions and liberation festival attendance (N = 1148)
Model 1
Model 2
Model 3
Model 4
Model 5
Model 6
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
ng intentions
ration festival attendance
.070**
.025
.061*
.024
.070**
.025
.068**
.025
.056*
.022
.050*
.021
ntal civic communication
.033***
.009
.019*
.008
enship in school
.016
.051
− .011
.035
enship outside school
.014
.042
− .007
.029
al sciences course
− .035
.032
− .022
.020
ent council
.039
.034
.017
.023
ntary work (ref. never)
casionally
.019
.025
− .002
.023
ucturally
.073**
.027
.048†
.026
cation (ref. vocational)
lege
.086**
.025
.079**
.025
versity
.13***
.024
.120***
.025
ntal ISEI score
.001
.001
.000
.001
ding (ref. never)
casionally
.027
.024
.018
.023
ucturally
− .026
.028
− .040
.028
eum (ref. never)
casionally
.048*
.023
.044†
.023
ucturally
.114***
.029
.101**
.032
ic group identification
− .036
.024
− .040†
.023
onal identification (ref. not)
rly strongly
.102*
.040
.099*
.039
y strongly
.105*
.041
.100*
.041
der (male)
.038†
.022
.030
.021
.035
.022
.035
.022
.034†
.020
.024
.021 M. Coopmans 630 le 2 (continued)
Model 1
Model 2
Model 3
Model 4
Model 5
Model 6
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
ng with parents
− .013
.025
− .010
.024
− .014
.025
− .015
.025
.002
.023
.001
.022
anisation
− .000
.001
− .000
.001
− .000
.001
− .000
.001
− .000
.000
− .000
.000
ration festival attendance
ntal civic communication
.039**
.012
.035**
.012
zenship in school
.001
.086
− .006
.077
zenship outside school
− .201**
.068
− .219***
.061
al sciences course
.010
.052
.021
.046
ent council
.110†
.057
.095†
.051
untary work (ref. never)
casionally
.128***
.032
.108**
.032
ucturally
.052
.040
.033
.041
cation (ref. vocational)
llege
.028
.043
.050
.040
iversity
− .014
.054
− .015
.050
ntal ISEI score
.000
.001
.000
.011
ding (ref. never)
casionally
.012
.037
− .009
.037
ucturally
.044
.039
.012
.039
eum (ref. 4.2 Explanatory Results The inclu-
sion of citizenship activities at school or involvement in voluntary organisations led to even
smaller changes.f Parental civic communication was found to positively affect both the likelihood of vot-
ing (an increased chance of 3%) and chances of visiting a festival (an increased chance of
4%), thereby providing partial support for Hypothesis 2, in which we hypothesised that
the positive association between young adults’ previous attendance at Liberation festivals
and their inclination to vote is partially explained by parental civic communication. We
found no evidence for the role of extra-curricular citizenship activities offered at school:
not in positively predicting either political or commemorative participation, nor in explain-
ing associations between previous festival attendance and voting intentions (Hypothesis 3). Contradicting our initial expectation, young adults who had attended schools organizing
democracy-related excursions were 20% less likely to have visited a Liberation festival. Stepwise analyses showed a similar picture for democracy-related in-school activities, such
as debates or mock elections. Furthermore, in line with expectations, young adults who
performed voluntary work were, on average, 7% more likely to vote, and 13% more likely
to attend a Liberation festival, thereby providing partial support for Hypothesis 4. In Model 5, we examined the extent to which educational track, parental socioeconomic
status, reading and museum activities, and ethnic and national identification explain the
found association between young adults’ previous festival attendance and their voting
intentions. A comparison with Model 1 revealed that, although the AME of Liberation fes-
tival attendance on voting inclinations in Model 5 was somewhat smaller (i.e. 6% instead
of 7%), the adjustment was minor, and the effect was still statistically significant. Young
adults who followed a college or university track had a 9 and 14% higher chance respec-
tively of voting in the next elections than youngsters with a vocational trajectory, yet did
not differ in their chances of having visited a Dutch Liberation festival, thereby providing
no support for Hypothesis 5a. Hypothesis 5b, on the role of parental socioeconomic status,
and Hypothesis 5c, on the role of reading activities, were not supported either, as none
of the effects were significant. 4.2 Explanatory Results never)
casionally
.064*
.032
.052
.032
ucturally
.110†
.057
.079
.060
nic group identification
− .052
.038
− .053
.038
onal identification (ref. not)
rly strongly
.161**
.051
.155**
.051 Dutch Liberation Festivals: A Vehicle to More Politically Active… 631 e 2 (continued)
Model 1
Model 2
Model 3
Model 4
Model 5
Model 6
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
y strongly
.173**
.052
.166**
.052
der (male)
− .051†
.029
− .058*
.029
− .046
.029
− .042
.029
− .036
.029
− .036
.029
ng with parents
− .060†
.033
− .056†
.033
− .059†
.033
− .056†
.033
− .059†
.033
− .049
.033
anisation
− .003*
.001
− .003*
.001
− .002†
.001
− .002*
.001
− .002*
.001
− .002†
.001
.10, *p < .05, **p < .01, ***p < .001
rage marginal effects for the outcome ‘yes, I would vote (vs. not)’ and ‘yes, I attended a Liberation festival at least once the past years (vs. never)’; b delta-method stand-
rrors Table 2 (continued)
Model 1
Model 2
Model 3
Model 4
Model 5
Model 6
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
dy/dxa
SEb
Very strongly
.173**
.052
.166**
.052
Gender (male)
− .051†
.029
− .058*
.029
− .046
.029
− .042
.029
− .036
.029
− .036
.029
Living with parents
− .060†
.033
− .056†
.033
− .059†
.033
− .056†
.033
− .059†
.033
− .049
.033
Urbanisation
− .003*
.001
− .003*
.001
− .002†
.001
− .002*
.001
− .002*
.001
− .002†
.001
† p < .10, *p < .05, **p < .01, ***p < .001
a Average marginal effects for the outcome ‘yes, I would vote (vs. not)’ and ‘yes, I attended a Liberation festival at least once the past years (vs. never)’; b delta-method stand-
ard errors 3 M. Coopmans 632 Liberation festival attendance on voting inclination when compared to Model 1: instead
of having a 7% higher chance of voting, youngsters who had visited a Liberation festival
over the past years were now shown to be 6% more likely to vote. However, the change was
small, and differences in voting inclinations between young adults who had never versus
those who had at least once attended a Liberation festival remained significant. 4.3 Additional Analyses To ensure that the found effect of previous Liberation festival attendance on voting inten-
tions did not differ across groups, additional analyses were conducted in which we included
interactions with our sociodemographic variables (i.e. educational trajectory, parental soci-
oeconomic status, reading and museum activities, and ethnic and national identification). The results of these analyses, which can be found in Table 4 in the “Appendix”, show that
the effect of festival attendance on voting intentions was not dependent on any of the soci-
odemographic characteristics included in this study. Moreover, to perform a stricter test of the potentially explanatory role of school in the
association between Liberation festival attendance and voting intentions, we conducted a
school-fixed-effects analysis, which controls for all characteristics of the school. This also
resulted in a larger sample (N = 2025), since also schools without information on their citi-
zenship activities could be included in the analysis. As Table 5 in the “Appendix” shows,
the positive effect of previous Liberation festival attendance on voting intentions remained
significant, indicating that the school environment does not explain this association. 4.2 Explanatory Results Young adults who more often visited museums, however,
were both more likely to vote (between 5 and 11%, depending on the frequency of activi-
ties), and 6% more likely to visit a Liberation festival, thereby providing partial support for
Hypothesis 5d.i National identification was also proven to be an important predictor of both past festi-
val attendance and future voting intentions. Youngsters who identified more strongly with
Dutch society had a 10 and 16% higher chance of voting and having attended a Libera-
tion festival respectively compared to those who did not identify with Dutch society. These
findings suggest evidence in support of Hypothesis 6b: The positive association between
young adults’ attendance at Dutch Liberation festivals and their inclination to vote can
be partially explained by their national identification. In Model 5, no effect was found of
ethnic identification. Stepwise analyses, however, indicated initially significant, negative
effects of ethnic identification on both voting intentions and festival attendance, which
disappeared after the addition of national identification. When not controlling for national
identification, young adults who identified with a non-Dutch ethnic group were 6 and 8%
less likely to vote and visit a Liberation festival, respectively. These findings thus provide
partial support for Hypothesis 6a. 633 Dutch Liberation Festivals: A Vehicle to More Politically Active… In Model 6, all alternative explanations were included simultaneously. Even though this
resulted in a further reduction of the AME of previous Liberation festival attendance on
future voting inclination, the effect was still significant: young people who had attended
a Liberation festival were 5% more likely to vote. All in all, our results thus do not pro-
vide proof of a spurious relationship between young people’s previous Liberation festival
attendance and their future voting intentions. 5 Conclusion and Discussion Moreo-
ver, festival attendance does not depend upon young adults’ educational track or their par-
ents’ socioeconomic status, suggesting that this type of informal commemoration, which
combines musical performances of famous artists with raising awareness of war, freedom,
and other core democratic rights and values, is a relatively inclusive form of commemora-
tion attracting a large, heterogeneous segment of the population—at least amongst youth. These findings are in line with earlier studies emphasising the attractiveness of ‘popular
culture’ elements amongst young citizens (Van Eijck and Knulst 2005), also in national
commemorations (Fricke 2013).l One aspect that does influence one’s chances of participating in commemorative activi-
ties is ethnic identification: young people who identify themselves with a non-Dutch eth-
nic group have lower chances of having visited a Liberation festival than young people
who do not identify with a non-Dutch ethnic group. The findings furthermore show that
this is explained by lower levels of national identification, i.e. they feel less like a member
of Dutch society. National identification was found to be an important predictor of both
voting intentions and festival attendance amongst all youngsters. These findings suggest
that Dutch Liberation festivals might place greater emphasis on the sufferings and heroism
of the Dutch rather than ‘core democratic values’, thereby failing to foster engagement in
the event amongst youth who identify less with Dutch society. This is similar to what was
found in an earlier study on commemorative participation amongst Dutch citizens with a
(non-Western) migration background (Coopmans et al. 2016), and highlights the impor-
tance of taking into account both ethnic and national identification when examining incli-
nations to participate in commemorative activities, also amongst young Dutch people with
grand- or great-grandparents born abroad. The civic home environment also plays a key role in young people’s commemorative
and political activities. The positive association that is found between Liberation festival
attendance and voting intentions is partially due to the more civically engaged home envi-
ronment. Young adults who more frequently discussed political and social issues with their
parents are not only more inclined to vote, but also have higher chances of having visited
a Liberation festival. Although youth participating in Liberation festivals appear to be a
heterogeneous crowd when it comes to educational and socioeconomic background, they
are thus more ‘selective’ where civic home environment is concerned. 5 Conclusion and Discussion To shed more light on the potential of (informal) national commemorations as motivators
of young people’s political behaviour, this article examined the extent to which 18-year-
olds’ previous participation in Dutch Liberation festivals is related to their intentions to
vote. Using structural equation modelling, we subsequently tested whether this relationship
was truly evidence of a motivating effect, or that it was in fact a spurious association. To do
so, we considered several alternative explanations of positive associations between young
people’s commemorative and political participation identified in previous studies as impor-
tant determinants of young citizens’ civic engagement. Our findings, however, show that,
even after taking into account the civic home environment, the civic school environment,
the out-of-school environment, and numerous sociodemographic factors, the difference in
voting chances between young adults who had never versus once or more visited a Libera-
tion festival remains statistically significant (i.e. young adults who have attended a Dutch
Liberation festival over the past years are around 5% more likely to be inclined to vote in
the next parliamentary elections), suggesting that there is indeed a motivating effect of this
particular type of commemoration on young people’s intentions to participate in political
activities. Although systematic empirical research on the consequences of commemorative par-
ticipation is limited, there have been some studies on the role of remembrance education,
including commemorative activities organized in the school context (Cowan and Maitles
2007, 2011). Based on comparisons of students’ attitudes and behaviours before and after
they studied the Holocaust and visited the Auschwitz-Birkenau Memorial Museum, these 1 1 3 3 M. Coopmans 634 studies conclude that remembrance education can promote young citizens’ civic attitudes
and behaviours by developing an understanding and knowledge of broader human rights
issues relating to historical and contemporary issues. The current study suggests that the
findings from Cowan and Maitles may also apply in a non-educational setting. The present
findings furthermore show that Dutch Liberation festivals are a popular form of commemo-
rating amongst youth: almost 60% has visited one at least once over the last years. 5 Conclusion and Discussion These findings not
only support previous studies on the vital role of parental communication in youth’s civic
socialisation processes (Hooghe and Boonen 2015; Jennings et al. 2009; Quintelier 2015b),
they also highlight that mnemonic socialisation, or, more specifically, the socialisation of
commemorative practices, is not restricted to communication about the historical events
that are the topic of these commemorations but instead can comprise a broader range of
civic issues. This conclusion is not only a valuable extension of mnemonic socialisation
theories, but also has important practical implications for those keen on promoting com-
memorative participation amongst young people. In addition to the civic home environment, leisure time activities outside the home envi-
ronment are also found to play a role in young people’s commemorative and political par-
ticipation. In line with previous research (Marzana et al. 2012; McFarland and Thomas
2006; Quintelier 2008, 2015a), youngsters who are more frequently involved in voluntary 1 3 Dutch Liberation Festivals: A Vehicle to More Politically Active… 635 organisations are not only more likely to vote, but are also more likely to visit Libera-
tion festivals. A similar picture is found for museum activities: young adults who more
frequently visit museums have also more often visited Liberation festivals and are more
inclined to vote. These findings support earlier studies on this topic (Cowan and Maitles
2011; Savenije and De Bruijn 2017). Moreover, they suggest that cultural resources provide
a better explanation for the association between young adults’ commemorative and politi-
cal participation than economic resources (see also Bourdieu 1984; Bourdieu and Passeron
1977), which is in line with the idea that cultural, commemorative, and political activities
are all part of a broader civic engagement (Adler and Goggin 2005; Amnå 2012). The civic school environment, in this study measured as the extra-curricular citizen-
ship activities offered by a school, was not found to influence the association between Lib-
eration festival attendance and voting intentions. Moreover, no effect was found on young
adults’ political engagement, which seems to contradict previous studies (although, admit-
tedly, the evidence so far has been mixed; Geboers et al. 2013). One explanation for this
lack of effect is that we have concentrated on the citizenship activities offered, not con-
sidering the student’s actual participation in the organised activities. 5 Conclusion and Discussion Even though volun-
tary extra-curricular citizenship activities at school have been found to have a more posi-
tive impact on young people’s citizenship than obligatory activities (Geboers et al. 2013),
this approach ignores within-school differences between students actively and less actively
participating in the organised activities. An alternative explanation is that other forms of
citizenship education, such as the pedagogical climate or curriculum in school, are more
effective in impacting adolescents’ future citizenship behaviours. School fixed effects mod-
els, however, indicated that the found association between commemorative and political
participation could not be explained by the school. This is in line with a recent study by
Dijkstra and colleagues (Dijkstra et al. 2015), in which it was concluded that citizenship
outcomes are better explained by factors at the student level than at the school level. An
interesting avenue for future research would therefore be to zoom in on the civic engage-
ment of and civic talks with peers, both inside and outside school, as they are an important
source of influence during adolescence (Brechwald and Prinstein 2011).if l
To be able to draw firmer conclusions regarding an actual motivating effect of young
people’s participation in informal commemorations on their political participation,
longitudinal research is needed to more reliably test the causality of the relationship
between the various activities. Moreover, a more comprehensive picture of the complete
civic and political socialisation process is needed, including a wider variety of political
attitudes and behaviours (see for instance Oser 2017). Ideally, we would follow young-
sters during different developmental phases of their civic socialisation, creating a data-
set with extensive information on changes in their political, social, and cultural activi-
ties, and including not only voting intentions but also actual voter turnout (even though
Hainmueller et al. (2015), for instance, quite convincingly demonstrate the close link
between hypothetical and actual voting choices). This would also enable us to examine
more dynamic processes such as the influence of changes in social environments (e.g. switching classes, schools, and going off to college), or interactions between different
social environments at different points in time (e.g. the changing role of parents versus
peers), as well as the underlying mechanisms that explain how participation in com-
memorative activities can lead to more political participation in later life. 5 Conclusion and Discussion In one of the national surveys conducted by the National Committee 4 and 5 May,
for instance, over 70% of the respondents indicated to use Dutch Remembrance Day and
Liberation Day to reflect on issues relating to (un)freedom, human rights, and democ-
racy (Verhue et al. 2017). Changes in awareness of and attitude on democratic rights and 3 M. Coopmans 636 values as a potential mediator could therefore be an interesting starting point. Another
interesting follow-up of the current research would be to track youngsters more closely
during their Liberation festival visits, to determine factors contributing to or disturbing
the politically motivating role of Dutch Liberation festivals. A mobile application could,
for instance, be used to register their activities and interactions with other festival visi-
tors. This would enable us to find out more about the concrete reasons for a potential
impact of attending Liberation festivals on young people’s political behaviours. What
happens during this event that makes people more interested or ready to participate? Is it the veterans’ stories, is it concrete political statements, or something else? Does
everybody who attends such an event also attend the politically relevant parts of it? A recent study by De Regt and Van der Lippe (2017) suggests otherwise. Answers to
these questions would allow for more in-depth analysis and are an interesting avenue for
future research.i All in all, however, the present findings provide tentative evidence of a positive
influence of previous participation in Dutch Liberation festivals on young adults’ vot-
ing intentions, thereby supporting Sapiro (2004) in her claim that the commemora-
tion of a national past should be considered a relevant aspect of political socialisation. Even though the effect is relatively small, the combination of popular culture elements,
shared moments, and references to past and present issues relating to war and freedom
used in Dutch Liberation festivals to emphasise the importance of core democratic val-
ues such as freedom and tolerance seems to motivate young visitors to contribute to the
continuance of the democratic system, at least when it comes to voting. Combined with
the fact that the Liberation festivals are a popular activity amongst young adults from
various socioeconomic backgrounds, and can therefore be considered a relatively inclu-
sive activity, this study shows that informal commemorations, such as the Dutch Libera-
tion festivals examined, have the potential to promote political participation amongst all
young people equally. 5 Conclusion and Discussion At the same time, Liberation festivals are less often attended by
youth identifying with a non-Dutch ethnic background—which is explained by lower
levels of national identification—thereby risking reinforcing gaps in political engage-
ment between youth with and without a migration background. Acknowledgements Earlier versions of this article were presented at the “CILS4EU 2015 conference” in
Stockholm, the “Migration and Social Stratification seminar” in Utrecht, and the “111th ASA Annual Meet-
ing” in Seattle. The author thanks participants in those sessions for their helpful comments and is especially
grateful for the insightful suggestions of Duane Alwin, Eva Jaspers, Tanja van der Lippe, Marcel Lubbers,
Miles Hewstone, Sabrina De Regt, Sara Geven, and Bas Hofstra on previous drafts of this paper. Funding CILS4EU was funded by the NORFACE ERA NET Plus Migration in Europe-programme. CILSNL is part of the research programme Investeringen Middelgroot MaGW with Project Number 480-
11-013, which is (partly) financed by the Netherlands Organisation for Scientific Research (NWO). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Inter-
national License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if changes were made. Appendix See Tables 3, 4 and 5. 1 3 Dutch Liberation Festivals: A Vehicle to More Politically Active… 637 ble 3 Datasets used for
able construction
Measurement
Datasets used
Voting intentions
w5_ym_nl_v5.0.0.dta
Liberation festival attendance
Involvement in voluntary organisations
Reading activities
Museum activities
Ethnic group identification
National identification
Gender
Living with parents
Parental civic communication
w2_ym_nl_v2.3.0.dta
w2_ym_nl_out_v2.3.0.dta
Citizenship activities outside school
CILSNL_citizenshipedu-
cation_v1.0.dta
Citizenship activities in school
Extra social sciences course
Student council
Urbanisation
Educational track
w1_ym_nl_v1.2.0.dta
w1_ym_nl_out_v1.0.0.dta
w2_ym_nl_v2.3.0.dta
w2_ym_nl_out_v2.3.0.dta
w3_ym_nl_v3.1.0.dta
w3_ym_nl_out_v3.1.0.dta
w4_ym_nl_v4.0.0.dta
w5_ym_nl_v5.0.0.dta
Parental ISEI score
w1_p_nl_v1.2.0.dta
w1_p_nl_out_v1.0.0.dta
w2_p_nl_out_v2.3.0.dta
w1_ym_nl_v1.2.0.dta
w1_ym_nl_out_v1.0.0.dta
w2_yn_nl_v2.3.0.dta
w2_yn_nl_out_v2.3.0.dta Table 3 Datasets used for
variable construction w2_ym_nl_v2.3.0.dta
w2_ym_nl_out_v2.3.0.dta
CILSNL_citizenshipedu-
cation_v1.0.dta Parental ISEI score 1 3 1 3 M. Coopmans 638 1 3
Table 4 Additional analyses, including interactions with sociodemographic variables (N = 1148)
Model 1
Model 2
Model 3
Model 4
Model 5
Model 6
Ba
SE
Ba
SE
Ba
SE
Ba
SE
Ba
SE
Ba
SE
Voting intentions
Constant
− .046
.508
− .081
.525
− .033
.515
− .149
.510
− .215
.502
− .166
.528
Liberation festival attendance
.354
.245
.372
.438
.300
.315
.482†
.262
.610**
.229
.568
.479
Parental civic communication
.193*
.079
.190*
.079
.195*
.079
.189*
.079
.184*
.079
.190*
.080
Citizenship in school
− .125
.348
− .111
.349
− .121
.348
− .130
.352
− .121
.349
− .104
.350
Citizenship outside school
− .073
.288
− .063
.289
− .074
.288
− .063
.291
− .064
.288
− .070
.290
Social sciences course
− .227
.205
− .223
.202
− .226
.201
− .216
.205
− .237
.201
− .224
.204
Student council
.162
.226
.171
.227
.160
.226
.159
.231
.169
.226
.174
.228
Voluntary work (ref. never)
Occasionally
− .019
.222
− .014
.222
− .008
.223
− .007
.223
− .019
.222
− .010
.222
Structurally
.533†
.320
.530†
.320
.531†
.320
.495
.321
.520
.320
.532†
.320
Education (ref. vocational)
College
.555†
.330
.712**
.240
.704**
.239
.749**
.242
.690**
.240
.718**
.241
University
1.114**
.400
1.307***
.313
1.334***
.314
1.313***
.315
1.296***
.313
1.300***
.314
Parental ISEI score
.004
.005
.003
.007
.004
.005
.005
.005
.005
.005
.005
.005
Reading (ref. never)
Occasionally
.194
.253
.192
.255
.211
.339
.209
.254
.203
.254
.197
.254
Structurally
− .366
.257
− .372
.257
− .614†
.334
− .370
.257
− .359
.257
− .376
.257
Museum (ref. never)
Occasionally
.420
.217
.422†
.217
.410†
.217
.346
.289
.436*
.218
.425†
.217
Structurally
1.216
.563
1.238*
.563
1.254*
.565
2.146*
1.066
1.268*
.563
1.255*
.564
Ethnic group identification
− .387†
.233
− .399†
.233
− .400†
.233
− .400†
.234
− .660*
.317
− .400†
.233
National identification (ref. 1 3 not)
Fairly strongly
.814**
.286
.814**
.286
.826**
.287
.846**
.289
.843**
.287
.909*
.369
Very strongly
.810**
.297
.818**
.296
.826**
.297
.840**
.299
.854**
.297
.793*
.374 Dutch Liberation Festivals: A Vehicle to More Politically Active… 639 Table 4 (continued)
Model 1
Model 2
Model 3
Model 4
Model 5
Model 6
Ba
SE
Ba
SE
Ba
SE
Ba
SE
Ba
SE
Ba
SE
Gender (male)
.249
.205
.243
.204
.240
.204
.251
.205
.254
.205
.245
.205
Living with parents
.001
.220
.010
.220
.000
.221
.000
.222
− .004
.221
.011
.221
Urbanisation
− .003
.004
− .003
.004
− .003
.004
− .003
.005
− .003
.004
− .003
.005
Interactions
Festival * education
Festiva * college
.319
.455
Festival * university
.438
.591
Festival * parental ISEI score
.003
.010
Festiva * reading
Festival * occasionally
− .013
.473
Festival * structurally
.493
.441
Festival * museum
Festival * occasionally
.144
.391
Festiva * structurally
− 1.493
1.240
Festival * ethnic identification
.508
.426
Festival * national identification
Festival * fairly strongly
− .214
.580
Festival * very strongly
.018
.569
† p < .10, *p < .05, **p < .01, ***p < .001
a Original coefficient estimates of the multilevel logistic generalised structural equation model are shown (coefficient estimates predicting festival attendance have been left out
to improve readability) 1 M. Coopmans 640 5 Additional analyses,
fixed effects model
ng voting intentions
25)
† p < .10, *p < .05, **p < .01, ***p < .001
a Original coefficient estimates of the conditional fixed-effects logistic
regression are shown
Ba
SE
Liberation festival attendance
.444**
.152
Parental civic communication
.233***
.056
Voluntary work (ref. never)
Occasionally
.200
.168
Structurally
.194
.208
Education (ref. vocational)
College
.725**
.248
University
.472
.383
Parental ISEI score
.007†
.004
Reading (ref. never)
Occasionally
.285
.181
Structurally
− .071
.188
Museum (ref. never)
Occasionally
.350*
.160
Structurally
1.001*
.387
Ethnic group identification
− .258
.181
National identification (ref. not)
Fairly strongly
.436*
.216
Very strongly
.757**
.227
Gender (male)
.319*
.149
Living with parents
.161
.160 Ba
SE
Liberation festival attendance
.444**
.152
Parental civic communication
.233***
.056
Voluntary work (ref. never)
Occasionally
.200
.168
Structurally
.194
.208
Education (ref. vocational)
College
.725**
.248
University
.472
.383
Parental ISEI score
.007†
.004
Reading (ref. never)
Occasionally
.285
.181
Structurally
− .071
.188
Museum (ref. 1 3 never)
Occasionally
.350*
.160
Structurally
1.001*
.387
Ethnic group identification
− .258
.181
National identification (ref. not)
Fairly strongly
.436*
.216
Very strongly
.757**
.227
Gender (male)
.319*
.149
Living with parents
.161
.160 Table 5 Additional analyses,
school fixed effects model
predicting voting intentions
(N = 2025) † p < .10, *p < .05, **p < .01, ***p < .001 a Original coefficient estimates of the conditional fixed-effects logistic
regression are shown a Original coefficient estimates of the conditional fixed-effects logistic
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English
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Cyclin-dependent Kinase 8/19 Inhibitor BCD 115
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Definitions
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cc-by
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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Cyclin-dependent Kinase 8/19 Inhibitor
BCD 115 National Cancer Institute Qeios ID: IXSTJP · https://doi.org/10.32388/IXSTJP Source National Cancer Institute. Cyclin-dependent Kinase 8/19 Inhibitor BCD 115. NCI
Thesaurus. Code C148166. National Cancer Institute. Cyclin-dependent Kinase 8/19 Inhibitor BCD 115. NCI
Thesaurus. Code C148166. An orally bioavailable inhibitor of cyclin dependent kinases 8 and 19 (CDK8/19), with
potential antineoplastic and chemoprotective activities. Upon oral administration,
CDK8/19 inhibitor BCD 115 binds to and inhibits the activity of CDK8/19, which prevents
activation of CDK8/19-mediated oncogenic signaling pathways, blocks selective
transcription of certain tumor promoting genes, and inhibits proliferation of CDK8/19-
overexpressing tumor cells. CDKs are serine/threonine kinases involved in the regulation
of the cell cycle and may be overexpressed in certain cancer cell types. CDK8 plays a key
role in transcription regulation and is an important oncogenic driver in a variety of cancer
cell types. 1/1
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Smooth Granular Sound Texture Synthesis by Control of Timbral Similarity
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Smooth Granular Sound Texture Synthesis by Control
of Timbral Similarity
Diemo Schwarz Sean O ’Leary Diemo Schwarz, Sean O ’Leary To cite this version: Diemo Schwarz, Sean O ’Leary. Smooth Granular Sound Texture Synthesis by Control of Timbral
Similarity. Sound and Music Computing (SMC), Jul 2015, Maynooth, Ireland. pp.6. hal-01182793 Distributed under a Creative Commons Attribution 4.0 International License 1. INTRODUCTION The synthesis of credible environmental sound textures
such as wind, rain, fire, crowds, traffic noise, is a crucial
component for many applications in computer games, in-
stallations, audiovisual production, cinema. Often, sound
textures are part of the soundscape of a long scene, and in
interactive applications such as games and installations, the
length of the scene is not determined in advance. There-
fore, it is advantageous to be able to play a given texture
for an arbitrary amount of time, but simple looping would
introduce repetition that is easy to pick out. Using very
long loops, or layering several loops can avoid this prob-
lem (and is the way sound designers currently do this), but
this stipulates that a long enough recording of a stable en-
vironmental texture is available, and uses up a lot of media
and memory space. Specifically, the present research draws on previous work
on corpus-based sound texture synthesis [20, 21], that can
also be seen as content-aware granular synthesis, and ex-
tends the work of Fr¨ojd and Horner [5] by the explicit mod-
eling and control of timbral similarity on randomised gran-
ular playback. Other methods to extend a given texture
are based on modeling of higher-order statistical proper-
ties [1, 9, 11, 12]. All these latter methods need a source
recording with stable and uniform texture content while
our proposed method can work with more varied textures
by being aware of the timbral content of all grains. We present here a method to extend an environmental
sound texture recording for an arbitrary amount of time,
without the need for the source recording to be of a stable
and uniform timbre or density. This means, a sound de-
signer can use a recording that fits the scene in atmosphere,
but without needing to isolate a stable and sufficiently long
loop, since our method will ensure smooth timbral tran-
sitions, while still varying the texture to avoid repetition
effects. Other methods for sound textures go further by model-
ing and recreating the typical transitions occurring in the
source texture by wavelet- or Markov-trees [3, 7, 8]. A recent approach by Heittola et al. [6] quite similar
to ours is aimed at full soundscape synthesis to recreate
the acoustic environment of a specific location for digi-
tal maps. There, the timbral similarity is calculated on
MFCCs and their deltas averaged over four second grains. Smooth Granular Sound Texture Synthesis by Control of Timbral Similarity Diemo Schwarz, Sean O’Leary
Ircam–CNRS–UPMC
firstname.secondname@ircam.fr Diemo Schwarz, Sean O’Leary
Ircam–CNRS–UPMC ABSTRACT Our method is based on randomised granular playback
with control of the similarity between grains using two dif-
ferent timbral distance measure that are compared in an
evaluation: a timbral distance based on audio descriptors,
and an MFCC-based distance. We also compare to purely
randomised playback as a baseline. Granular methods to synthesise environmental sound tex-
tures (e.g. rain, wind, fire, traffic, crowds) preserve the
richness and nuances of actual recordings, but need a
preselection of timbrally stable source excerpts to avoid
unnaturally-sounding jumps in sound character. To over-
come this limitation, we add a description of the timbral
content of each sound grain to choose successive grains
from similar regions of the timbre space. We define two
different timbre similarity measures, one based on per-
ceptual sound descriptors, and one based on MFCCs. A
listening test compared these two distances to an uncon-
strained random grain choice as baseline and showed that
the descriptor-based distance was rated as most natural, the
MFCC based distance generally as less natural, and the
random selection always worst. 2. PREVIOUS AND RELATED WORK The method presented here situates itself in the granular
synthesis-based approaches to sound textures, as opposed
to ones based on signal or physical models. These meth-
ods need a recording as source material from which sound
grains are picked and played back. Granular playback
takes advantage of the richness of actual recorded sound,
in contrast to other methods based on pure synthesis [4],
see the state-of-the-art overview on sound texture synthe-
sis [19] for further discussion and a general introduction
of sound textures. Fr¨ojd and Horner [5] use purely ran-
domised playback of long grains (around one second), with
half-grain cross-fade, and slight randomisation of play-
back parameters (detuning, amplification) to avoid repe-
tition. O’Leary and R¨obel’s Montage approach [14, 15]
exchanges grains by timbral similarity to avoid repetition,
while following template sequences from the original, and
introduce a spectral cross-fade minimising phase distor-
tion. HAL Id: hal-01182793
https://hal.science/hal-01182793v1
Submitted on 3 Aug 2015 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Copyright: c⃝2015 Diemo Schwarz et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution 3.0 Unported License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited. 3. TEXTURE SYNTHESIS 4. The successor grain s is chosen randomly from the
c candidate grains. If s is within one second of q, a
new grain s is picked from the candidates, to avoid
picking grains too close to each other. 4. The successor grain s is chosen randomly from the
c candidate grains. If s is within one second of q, a
new grain s is picked from the candidates, to avoid
picking grains too close to each other. Our method is derived from corpus-based concatenative
synthesis (CBCS) [18], where grains are played back from
a corpus of segmented and descriptor-annotated sounds. a corpus of segmented and descriptor annotated sounds. Usually, CBCS is used to control the timbral evolution of
the synthesised sound while still using original recordings
as the sound source. This can be applied to texture syn-
thesis to match the sound to the evolution of a given scene
[20], see also the example video of interactive wind texture
synthesis in a 2D descriptor space 1 , when the descriptor
target is given directly by the sound designer, or by the
game engine. However, in the application described here,
we don’t want to control the timbral output directly, but
have the system synthesise a varying texture without audi-
ble repetitions nor artefacts such as abrupt timbral or loud-
ness changes. To this end, we use a timbral distance mea-
sure d between the last played grain and all other grains
as candidates, and randomly select a successor grain from
the timbrally closest grains, thus generating a random walk
through the timbral space of the recording, that never takes
too far a step, but that potentially still traverses the whole
space of expression of the recording. 5. To avoid too regular triggering of new grains, the du-
ration and time of the next grain are randomly drawn
within a 600–1000 ms range, and a random start off-
set of +/- 200 ms is applied to each grain. 6. Played grains are overlapped by 200 ms, and an
equal-power sinusoidal cross-fade is applied during
the overlap. 7. While the desired length of the output texture is not
reached, the chosen grain s becomes the query grain
q, and the algorithm continues at step 3. 1 http://imtr.ircam.fr/imtr/Sound Texture Synthesis 1. INTRODUCTION The resulting similarity matrix serves to coalesce adjacent
grains into longer segments, and to cluster these in order to
control the smoothness of transitions. Copyright: c⃝2015 Diemo Schwarz et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution 3.0 Unported License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited. 4. RESULTS AND EVALUATION 1. We construct a corpus of one or more recordings,
segment it into grains (here of length 800 ms with-
out overlap), and analyse each grain i for its timbral
characteristics in a feature vector ui. The method presented here is evaluated in an ongoing lis-
tening test accessible online. 3 At the time of writing, 31
subjects took the test. The test consists of a questionnaire with 7 second ex-
tracts of 7 sound examples listed in table 1. This small
test database contains sounds from [3] that are widely
used in evaluation of sound textures [5, 10] and thus par-
tially allows comparison of the results. Other sounds were
contributed by [20] and by the partners of the PHYSIS
project. 4
All sounds were chosen for their properties of
being a non-uniform environmental sound texture, i.e. con-
taining some variation in texture and timbre, but not clearly
distinguishable short sound events. An exception is the
Baby Crying sound, that is here as an extreme counterex-
ample, since it contains very different and well-separated
cries. In our experiments we used two variants of annota-
tion giving rise to two different distance measures: In our experiments we used two variants of annota-
tion giving rise to two different distance measures: (a) An analysis of the 7 audio descriptors validated
by [20], extracted with the IRCAMDESCRIP-
TOR library [16]: The mean of the instanta-
neous descriptors Loudness, FundamentalFre-
quency, Noisiness, SpectralCentroid, Spectral-
Spread, SpectralSlope over all frames of size
23 ms. (b) An analysis of the timbral shape in terms of
the mean of the mel-frequency cepstral coeffi-
cients (MFCCs) over the segment. Sound Example
Description
Lapping Waves
long-term structure
Desert Wind
wind with occasional gusts
Stadium Crowd
atmosphere, occasional cheering
and honking
Water Faucet
various speeds of water flow
Formula One
not actually a texture, containing
structured variation
Traffic Jam
motor sounds, honking, some
shouts
Baby Crying
not actually a texture, containing
large variation
Table 1. List of test sound database and description Sound Example
Description
Lapping Waves
long-term structure
Desert Wind
wind with occasional gusts
Stadium Crowd
atmosphere, occasional cheering
and honking
Water Faucet
various speeds of water flow
Formula One
not actually a texture, containing
structured variation
Traffic Jam
motor sounds, honking, some
shouts
Baby Crying
not actually a texture, containing
large variation
Table 1. List of test sound database and description 2. 3.1 Implementation The prototype system is implemented in Max/MSP us-
ing the MuBu (Multi-Buffer) extension library [17],
with the integration of the batch analysis module
pipo.ircamdescriptor. 2 The algorithm proceeds as follows: 2 http://forumnet.ircam.fr/product/max-sound-box
3 http://ismm.ircam.fr/sound-texture-transition-control-evaluation
4 http://sites.google.com/site/physisproject p
4 http://sites.google.com/site/physisproject 3 http://ismm.ircam.fr/sound-texture-transition-control-evaluation 4. RESULTS AND EVALUATION Box plot of the scaled naturalness ratings
per type of stimulus, showing the median (middle line),
quartile range (box), min/max (whiskers), and outliers
(crosses). orig
descr
mfcc
random
Naturalness
0
10
20
30
40
50
60
70
80
90
100 For each example, the original, and 4 test stimuli of 7 s
length are presented. The stimuli contain in randomised
order the 3 syntheses (by descriptor distance, MFCC dis-
tance, random), and the original as hidden reference. For
each stimulus, the subject is asked to rate the aspect of
Naturalness on a scale of 0–100, with labels given in ta-
ble 2. Note that the question of Sound Quality does not
make sense for this evaluation since no signal processing
other than long cross-fades is applied, and therefore the
perceived sound quality is the same for all stimuli. We linearly scaled the collected naturalness ratings indi-
vidually for each subject (over all sounds) to a range of 0
to 100. The rationale is that the relative ratings of overly
enthusiastic or overly critical subjects are thus made com-
parable with the rest of the subjects. We can see in figures 3
and 4 that only a few subjects (notably 1, 12, 14, 27) ex-
hibit very narrow rating ranges. Figure 1. Box plot of the scaled naturalness ratings
per type of stimulus, showing the median (middle line),
quartile range (box), min/max (whiskers), and outliers
(crosses). The collected data is summarised in figure 1. We can
see that the descriptor-based similarity measure generally
obtains better ratings than the MFCC based one, that the
random grain choice is rated worst, and that the originals
are rated very high, with only a few outliers. that only for Stadium Crowd and Water Faucet, and to
a lesser degree for Formula One, descriptor and MFCC-
based distance are rated significantly different. We hypoth-
esise that especially these sounds benefit greatly from the
more detailed descriptors Loudness and FundamentalFre-
quency, as they contain sequences of pitched foreground
events. For all sounds but Traffic Jam and Baby Crying the
descriptor-based distance is significantly rated better than
the random method, while for the MFCC-based distance
this is only so for Lapping Waves and Desert Wind. 4. RESULTS AND EVALUATION For synthesis, we start with a seed grain q, selected
randomly or given manually to start off with a cer-
tain timbral content. 3. When a grain is triggered, c = 5 successor grains
are searched by a (c + 1)-nearest neighbour search,
i.e., given the current grain’s descriptor values uq
as query point, the kD-tree [2, 22] finds in logarith-
mic time the c candidate grains with descriptor val-
ues closest to the query (and the query grain q itself,
since it has a distance of zero). The distance func-
tion is a weighted Euclidean distance, with weights
given by the inverse standard deviation to normalise
the search space. Multiplying the weights allows us
further to give more importance to certain descrip-
tors, or to exclude them from influencing the search. 3. When a grain is triggered, c = 5 successor grains
are searched by a (c + 1)-nearest neighbour search,
i.e., given the current grain’s descriptor values uq
as query point, the kD-tree [2, 22] finds in logarith-
mic time the c candidate grains with descriptor val-
ues closest to the query (and the query grain q itself,
since it has a distance of zero). The distance func-
tion is a weighted Euclidean distance, with weights
given by the inverse standard deviation to normalise
the search space. Multiplying the weights allows us
further to give more importance to certain descrip-
tors, or to exclude them from influencing the search. orig
descr
mfcc
random
Lapping Waves
85.09 (± 20.70)
73.04 (± 19.76)
71.82 (± 23.58)
46.01 (± 25.16)
Desert Wind
92.46 (± 08.70)
59.90 (± 22.28)
61.97 (± 26.19)
23.24 (± 23.11)
Stadium Crowd
91.65 (± 13.36)
56.22 (± 29.05)
23.03 (± 18.52)
25.83 (± 20.18)
Water Faucet
86.82 (± 16.93)
55.38 (± 24.01)
25.34 (± 18.11)
14.18 (± 15.11)
Formula One
95.15 (± 08.57)
29.55 (± 20.62)
17.43 (± 19.61)
12.85 (± 15.71)
Traffic Jam
77.36 (± 26.44)
59.01 (± 31.47)
56.23 (± 29.07)
52.97 (± 27.07)
Baby
95.43 (± 09.45)
17.98 (± 15.15)
13.07 (± 15.38)
15.89 (± 21.02)
Total
89.14 (± 17.30)
50.16 (± 29.76)
38.41 (± 31.37)
27.28 (± 26.15)
Table 3. Naturalness rating mean and standard deviation over all subjects. Table 3. Naturalness rating mean and standard deviation over all subjects. orig
descr
mfcc
random
Naturalness
0
10
20
30
40
50
60
70
80
90
100
Figure 1. 4. RESULTS AND EVALUATION An-
other remark is that for Lapping Waves and Traffic Jam
the original is not rated significantly different from the
descriptor-based method, and for the former sound this
also applies to the MFCC-based method. To test if the observed differences of means are signif-
icant or simply due to chance, further statistical analysis
has been carried out using the ANOVA (analysis of vari-
ance) method with Bonferroni correction. Here the null
hypothesis H0 is that means are equal, and differences are
due to chance, and the alternative hypothesis HA is that the
means are not equal. The p-values and significance levels 5
for each pair of comparisons are given in tables 4 and 5 for
the raw and scaled ratings, respectively. The scaling seems
to augment the contrast of the results, leading to a rise in
significance level for a few pairs in the ANOVA results. ANOVA confirms that globally the descriptor-based sim-
ilarity is preferred over MFCC, and both are preferred over
the random method. However, the detailed analysis shows 5. CONCLUSIONS AND FUTURE WORK Score
Label
0-19
Very unnatural: repetitions, jumps, cuts. 20-39
Somewhat unnatural
40-59
Somewhat natural
60-79
Very natural
80-100
As natural as original
Table 2. Naturalness rating scale Score
Label
0-19
Very unnatural: repetitions, jumps, cuts. 20-39
Somewhat unnatural
40-59
Somewhat natural
60-79
Very natural
80-100
As natural as original
Table 2. Naturalness rating scale A possible explanation for the general superiority of the
descriptor-based similarity measure over the MFCC based
one is that the perceptual descriptors better capture cer-
tain aspects of the sound character of environmental tex-
tures beyond pure spectral shape (that is represented by
MFCCs). We can hypothesise that some of this informa-
tion is related to pitch content, as expressed by the Fun-
damentalFrequency and Noisiness descriptors. More re- Lapping Waves
Desert Wind
Stadium Crowd
Water Faucet
Formula One
Traffic Jam
Baby
0
10
20
30
40
50
60
70
80
90
100
Figure 2. Box plot of the scaled naturalness ratings per source sound and type of stimulus (orig, descr, mfcc, random). Lapping Waves
Desert Wind
Stadium Crowd
Water Faucet
Formula One
Traffic Jam
Baby
0
10
20
30
40
50
60
70
80
90
100
Figure 2. Box plot of the scaled naturalness ratings per source sound and type of stimulus (orig, descr, mfcc, random). Figure 2. Box plot of the scaled naturalness ratings per source sound and type of stimulus (orig, descr, mfcc, random). Subject No. 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
0
20
40
60
80
100
Figure 3. Box and dot plot of the per-subject naturalness rating prior to scaling (◦original, ⋄descr, ▽mfcc, × random). Subject No. 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
0
20
40
60
80
100
Figure 3. Box and dot plot of the per-subject naturalness rating prior to scaling (◦original, ⋄descr, ▽mfcc, × random). Subject No. 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
0
20
40
60
Figure 3. 5. CONCLUSIONS AND FUTURE WORK orig
orig
orig
descr
descr
mfcc
descr
mfcc
random
mfcc
random
random
Lapping Waves
0.1772
0.2037
0.0000 ****
1.0000
0.0003 ***
0.0002 ***
Desert Wind
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
0.0000 ****
0.0000 ****
Stadium Crowd
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
Water Faucet
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.1179
Formula One
0.0000 ****
0.0000 ****
0.0000 ****
0.1576
0.0106 *
1.0000
Traffic Jam
0.0833
0.0338 *
0.0115 *
1.0000
1.0000
1.0000
Baby
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
1.0000
1.0000
total
0.0000 ****
0.0000 ****
0.0000 ****
0.0002 ***
0.0000 ****
0.0002 ***
Table 4. P-values and significance class 5 for each pair of differences of means on unscaled naturalness ratings. orig
orig
orig
descr
descr
mfcc
descr
mfcc
random
mfcc
random
random
Lapping Waves
0.2360
0.1409
0.0000 ****
1.0000
0.0000 ****
0.0001 ***
Desert Wind
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
0.0000 ****
0.0000 ****
Stadium Crowd
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
Water Faucet
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.1408
Formula One
0.0000 ****
0.0000 ****
0.0000 ****
0.0365 *
0.0012 **
1.0000
Traffic Jam
0.0858
0.0297 *
0.0075 **
1.0000
1.0000
1.0000
Baby
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
1.0000
1.0000
total
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0001 ****
Table 5. P-values and significance class 5 for each pair of differences of means on scaled naturalness ratings. P-values and significance class 5 for each pair of differences of means on unscaled naturalness ratings. Table 4. P-values and significance class 5 for each pair of differences of means on unscaled naturalness ratings. orig
orig
orig
descr
descr
mfcc
descr
mfcc
random
mfcc
random
random
Lapping Waves
0.2360
0.1409
0.0000 ****
1.0000
0.0000 ****
0.0001 ***
Desert Wind
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
0.0000 ****
0.0000 ****
Stadium Crowd
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
Water Faucet
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.1408
Formula One
0.0000 ****
0.0000 ****
0.0000 ****
0.0365 *
0.0012 **
1.0000
Traffic Jam
0.0858
0.0297 *
0.0075 **
1.0000
1.0000
1.0000
Baby
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
1.0000
1.0000
total
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0001 ****
Table 5. 5. CONCLUSIONS AND FUTURE WORK P-values and significance class 5 for each pair of differences of means on scaled naturalness ratings. P-values and significance class 5 for each pair of differences of means on scaled naturalness ratings. search is necessary to test this hypothesis and to link it with
recent findings about fundamental mechanisms of sound
texture perception [13]. ence and possible automatic estimation of the neighbour-
hood parameter (the number of candidates c). To conclude, we hope that this method can improve
the workflow of sound designers for interactive or post-
production applications, and further augment the advan-
tages that procedural audio has to offer over fixed media in
order to foster uptake by the industry. While the presented method is not a sequence model that
tries to model and generate the temporality of variation
given an environmental recording, it can regenerate at least
some of the naturally occurring variation in texture record-
ings. This has the two advantages of having a more var-
ied output for background atmosphere sounds that uses the
whole range of sound occurring in a source recording, and
that the sound designer does not have to limit herself to sta-
ble textural recordings, or has to hunt down long-enough
stretches of stable texture in longer recordings. 5. CONCLUSIONS AND FUTURE WORK Box and dot plot of the per-subject naturalness rating prior to scaling (◦original, ⋄descr, ▽mfcc, × random). Subject No. 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
0
20
40
60
80
100
Figure 4. Box and dot plot of the per-subject naturalness rating after scaling (◦original, ⋄descr, ▽mfcc, × random). Figure 3. Box and dot plot of the per-subject naturalness rating prior to scaling (◦original, ⋄descr, ▽mfcc, × random). Subject No. 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
0
20
40
60
80
100
Figure 4. Box and dot plot of the per-subject naturalness rating after scaling (◦original, ⋄descr, ▽mfcc, × random). Subject No. 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
0
20
40
60
80
100 Figure 4. Box and dot plot of the per-subject naturalness rating after scaling (◦original, ⋄descr, ▽mfcc, × random). orig
orig
orig
descr
descr
mfcc
descr
mfcc
random
mfcc
random
random
Lapping Waves
0.1772
0.2037
0.0000 ****
1.0000
0.0003 ***
0.0002 ***
Desert Wind
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
0.0000 ****
0.0000 ****
Stadium Crowd
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
Water Faucet
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.0000 ****
0.1179
Formula One
0.0000 ****
0.0000 ****
0.0000 ****
0.1576
0.0106 *
1.0000
Traffic Jam
0.0833
0.0338 *
0.0115 *
1.0000
1.0000
1.0000
Baby
0.0000 ****
0.0000 ****
0.0000 ****
1.0000
1.0000
1.0000
total
0.0000 ****
0.0000 ****
0.0000 ****
0.0002 ***
0.0000 ****
0.0002 ***
Table 4. P-values and significance class 5 for each pair of differences of means on unscaled naturalness ratings. Acknowledgments The work presented here is partially funded by the French
Agence Nationale de la Recherche (ANR) within the
project PHYSIS, ANR-12-CORD-0006. The authors wish
to thank Wei-Hsiang Liao for his groundwork on the online
evaluation questionnaire, Axel R¨obel, the PHYSIS project
partners, and the Analysis–Synthesis and ISMM teams at
Ircam. Although this is not the topic of this article, synthesis can
be started off at specific-sounding grains in the recording
as seeds, in order to start the texture with a given atmo-
sphere (e.g. start with calm wind to not startle the listener
at the beginning of a new scene with a gust of wind). This
could, for instance, be achieved using a scatter-plot inter-
face that allows to visualise the timbral space in 2D as
popularised by the CATART software. 6 With a little fu-
ture work, the texture could then be made to move towards
another type of sound by specifying its feature vector and
favouring transitions that move towards that point in the
descriptor space. More future work should check the influ- 5 The significance level depending on the p-value is habitually repre-
sented by a number of stars as follows:
Level
*
**
***
****
p ≤
0.05
0.01
0.001
0.0001
6 h
//i
i
f / 5 The significance level depending on the p-value is habitually repre-
sented by a number of stars as follows: References [1] Joan Bruna and St´ephane Mallat. Audio Texture Syn-
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SI Materials and Methods Cell Lines and Materials. All cell lines were maintained in RPMI 1640 (GIBCO) supplemented with 5%
FBS (Omega Scientific), 1% penicillin/streptomycin, and 100 mM L-glutamine (Invitrogen). The
melphalan resistant cells, 8226/LR5 and U266/LR6, were passaged weekly in medium containing 5 PM or
6 PM melphalan, respectively. The following drugs and antibodies were used in these studies (sources in parentheses): melphalan (Sigma
Aldrich); BMS-345541 (Calbiochem); bortezomib (Millennium); D-phospho-IKKD/IKKE, D-IKKD, and D-
IKKE (Cell Signaling); D-E-Actin (Sigma); normal rabbit IgG (Upstate Cell Signaling); D-p65, D-p50, D-c-
Rel, D-RelB, and D-p52 (Santa Cruz Biotechnology); and D-FANCD2 (Novus Biologicals). The phospho-
IKKD/IKKE antibody specifically recognizes IKKD and IKKE after phosphorylation at Ser-176/Ser-180
and Ser-177/Ser-181, respectively. Electrophoretic Mobility Shift Assays (EMSA). In brief, NF-NB DNA binding activity was measured
using
a
probe
containing
the
well-described
sequence
of
the
NB
enhancer,
5’-
TAGTTGAGGGGACTTTCCCAG-3’. When indicated, nuclear extracts were incubated with the following
oligonucleotides
containing
FANCD2-specific
consensus
NF-NB
binding
sites:
5’-
TTCAGACAGGGGCTCTCCCATTGCAA-3’ (Probe I); 5’-TTTCCCCAGGAAACCCCAATTTGCAA-3’
(Probe
II);
5’-TTAATATACTAAAAAACCCTGAATAA-3’
(Probe
III);
and,
5’-
TTTGAAGTGGGGCTTCCCAGACTGAA-3’ (Probe IV). NF-1 DNA binding activity was used as a
control and was measured using a probe derived from the adenovirus-2 origin of replication (5’-
CTTATTTTGGATTGAAGCCAATAT-3’). The 5’ overhangs of these probes were filled in with Klenow
(exo-) in the presence of [D32P]ATP for 30 min at 37qC. Unincorporated nucleotide was removed by a gel
filtration column (Roche). 20-40 kcpm of probe was incubated with 5 Pg protein extract and 1Pg poly
dI:dC for 20 min at room temperature. Protein-DNA complexes were resolved on a 5% nondenaturating
polyacrylamide gel and detected by autoradiography. Transfection of siRNA. Transfection of siRNAs was performed as described previously (1). The following
siRNA sequences were used (target mRNAs in parentheses): 5’-UGGUUUACAUGUCGACUAA-3’, 5’-
UGGUUUACAUGUUGUGUGA-3’,
5’-UGGUUUACAUGUUUUCUGA-3’,
and
5’-
UGGUUUACAUGUUUUCCUA-3’
(siCONTROL,
Non-Targeting
pool);
5’-
CGUACGCGGAAUACUUCGA -3’ (Luciferase); 5’-GUGCCAGGAUCACGUAGAAUU-3’ (c-Rel); 5’-
CGAACAGCCUUGCAUCUAGUU-3’ (p52); 5’-GGAUUGAGGAGAAACGUAAUU-3’ (p65); 5’-
CUGCGGAUUUGCCGAAUUAUU-3’ (RelB); 5’-GGAGACAUCCUUCCGCAAAUU-3’ (p50); and, 5’-
UGGAUAAGUUGUCGUCUAU-3’,
5’-CAACAUACCUCGACUCAUU-3’,
5’- ,
,
GGAUUUACCUGUGAUAAUA-3’, 5’-GGAGAUUGAUGGUCUACUA-3’ (FANCD2 Targeting pool). Cells were typically used in experiments after 24-48 h of siRNA treatment. Chromatin Immunoprecipitation. The ChIP DNA was subjected to Real Time Quantitative PCR (qPCR)
using
the
following
primer
pairs:
5’-AATGAATGGGCAGCCGTTA-3’
and
5’-
TAGCCTCGCTCCACCTGACT-3’
(GAPDH,
control
samples);
5’-
GGGTCTGCAGGAGATCAACTAAGAAA-3’
and
5’-GTGCCTGGCCCTATGCTGTAACTA-3’
(FANCD2,
STATx);
and,
5’-GAGCCAAGAGGTACCCTGATAAAGTC-3’
and
5’-
CAGCTTTGGTTTAATACCTGTCAGAATT-3’ (FANCD2, NF-NB). Real-time Quantitative RT-PCR Analysis. SI Materials and Methods Fifty images were randomly captured per slide, and the logarithmic
radius of each nucleoid was calculated using the tail-length parameter of the Loats Associates comet
analysis software. y
DNA damage was analyzed using the Alkaline Comet Assay as previously described (2). 8226/LR5 cells
were either pre-treated with 3 nM bortezomib or control for 8 h followed by 5 h treatment with 50 PM
melphalan; or, transfected with the indicated siRNA duplexes, and, 48 h later, the cells were exposed to
various amounts of melphalan or vehicle control for 5 h. After drug treatment, cells were treated with 9 Gy
ionizing radiation (IR) and the Alkaline Comet assay was performed according to the manufacturer’s
protocol. The comet moment was scored for 50 comets from each cell line using Loats Associates comet
analysis software, and the percentage of cross-linking was calculated as follows: {1 [(comet moment drug
treated comet moment control)/(comet moment 9 Gy comet moment control)] } u 100. In both assays,
results were obtained from three independent trials. Statistical Analysis. To examine the effect of the loss of RelB/p50 on re-sensitization of 8226/LR5, a
linear regression was used to compare differences in melphalan-induced ICL in 8226/S and 8226/LR5
siRNA-transfected cells (i.e., LR5 RelB/p50 and LR5 siControl) relative to the 8226/LR5 wild-type cell
line. Also, analysis of variance (ANOVA) was employed to test whether nuclear DNA diffusion after
melphalan treatment was different among all experimental groups. The Tukey’s Honest Significance
Difference method was used to adjust P-values for multiple comparisons (3). To examine the relationship
between the nuclear DNA diffusion and ICL, Pearson correlation was used to study their association (4). T
h
ff
f BMS 345541
d b
ib
FA/BRCA
i
i
d
h h Statistical Analysis. To examine the effect of the loss of RelB/p50 on re-sensitization of 8226/LR5, a
linear regression was used to compare differences in melphalan-induced ICL in 8226/S and 8226/LR5
siRNA-transfected cells (i.e., LR5 RelB/p50 and LR5 siControl) relative to the 8226/LR5 wild-type cell
line. Also, analysis of variance (ANOVA) was employed to test whether nuclear DNA diffusion after
melphalan treatment was different among all experimental groups. The Tukey’s Honest Significance
Difference method was used to adjust P-values for multiple comparisons (3). To examine the relationship
between the nuclear DNA diffusion and ICL, Pearson correlation was used to study their association (4). SI Materials and Methods Initially, a customized microfluidic card (Applied
Biosystems) was used to simultaneously analyze the expression of 11 FA/BRCA related genes, brca1,
brca2, fanca, fancc, fancd2, fance, fancf, fancg, fancl, rad51, and rad51c, in 8226 cells. In later studies, and
in order to incorporate recent additions to the FA/BRCA family of genes, expression profiles were
determined for 15 FA/BRCA related genes, brca1, brca2, fanca, fancb, fancc, fancd2, fance, fancf, fancg,
fanci, fancj, fancl, fancm, fancn, and usp1, in U266 cells. For melphalan and bortezomib combination
studies, FANCD2 gene expression was determined using the FANCD2 20X Assay on Demand probe
(Applied Biosystems). Quantitative RT-PCR was carried out using the ABI 7900 Sequence Detection System, and results were analyzed using the comparative CT method of analysis and SDS 2.1 software
(Applied Biosystems). Samples were normalized to GAPDH, an endogenous control. System, and results were analyzed using the comparative CT method of analysis and SDS 2.1 software
(Applied Biosystems). Samples were normalized to GAPDH, an endogenous control. Immunofluorescence Analysis. Cells were treated with either vehicle control or bortezomib for 8 h,
followed by 5 h treatment with melphalan. A cytospin was performed using 60,000 cells/slide, followed by
fixation with 2% paraformaldehyde (Sigma). Slides were washed twice in PBS and cells were
permeabilized using 0.3% Triton X-100 in PBS supplemented with 1% normal goat serum (NGS; Sigma). Following permeabilization, cells were blocked with 5% NGS, 0.1% Triton X-100 in PBS. Slides were then
washed three times in PBS and stained with FANCD2 antibody for 2 h. Next, cells were washed three
times in PBS and Alexa Fluor 488 secondary antibody (Molecular Probes) was then added. DAPI (Sigma)
was used to visualize the nucleus. Fluorescence was detected using the Zeiss Axiovert Upright Fluorescent
Microscope, and 100 cells per condition (control; melphalan; bortezomib; and bortezomib plus melphalan)
were scored as having greater or less than 5 foci. Diffusion Apoptosis Slide Halo (DASH) and Alkaline Comet Assays. The DASH assay captures and
detects damage-induced low molecular-weight DNA fragments as diffuse halos in an agarose microgel. After the indicated siRNA transfections, the cells were treated with 25-100 PM melphalan or vehicle
control for 24 h, then washed with PBS and embedded in low melting agarose. The embedded cells were
lysed for 10 min under alkaline conditions, and DNA was precipitated and visualized with SYBR Green
fluorogenic dye (Molecular Probes). SI Materials and Methods To test the effect of BMS-345541 and bortezomib on FA/BRCA gene expression, we examined whether
fold change of gene expression was deviated away from 1 for each cell line (e.g., 8226/S, 8226/LR5, U266,
and U266/LR6). A fold change less than 1 indicates under-expression (compared to the vehicle control). Since each gene showed a non-linear pattern of fold change over time (see Figure 4), instead of using a
linear slope for testing, a linear model with time as a categorical explanatory variable was used to test the
null hypothesis of fold change=1 at each time point. Fold change differences between 8 and 12 h post-
BMS-345541 treatment in U266 and U226/LR6 cells using the paired t-test were also analyzed. Since we
tested multiple genes related to the FA/BRCA pathway, we adjusted p value based on the false discovery Statistical Analysis. To examine the effect of the loss of RelB/p50 on re-sensitization of 8226/LR5, a
linear regression was used to compare differences in melphalan-induced ICL in 8226/S and 8226/LR5
siRNA-transfected cells (i.e., LR5 RelB/p50 and LR5 siControl) relative to the 8226/LR5 wild-type cell
line. Also, analysis of variance (ANOVA) was employed to test whether nuclear DNA diffusion after
melphalan treatment was different among all experimental groups. The Tukey’s Honest Significance
Difference method was used to adjust P-values for multiple comparisons (3). To examine the relationship
between the nuclear DNA diffusion and ICL, Pearson correlation was used to study their association (4). To test the effect of BMS-345541 and bortezomib on FA/BRCA gene expression, we examined whether
fold change of gene expression was deviated away from 1 for each cell line (e.g., 8226/S, 8226/LR5, U266,
and U266/LR6). A fold change less than 1 indicates under-expression (compared to the vehicle control). Since each gene showed a non linear pattern of fold change over time (see Figure 4) instead of using a testing to 1. van den Hoff, M. J., A. F. Moorman, et al. Electroporation in 'intracellular' buffer increases cell survival.
Nucleic Acids Res 1992;20:2902.
2. Hazlehurst, L. A., S. A. Enkemann, et al. Genotypic and phenotypic comparisons of de novo and
acquired melphalan resistance in an isogenic multiple myeloma cell line model. Cancer Res. 2003;
63:7900-7906. ;
2. Hazlehurst, L. A., S. A. Enkemann, et al. Genotypic and phenotypic comparisons of de novo and
acquired melphalan resistance in an isogenic multiple myeloma cell line model. Cancer Res. 2003;
63:7900-7906. y
g p p
;
5. Benjamini, Y., and Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach
to multiple testing. Journal of the Royal Statistical Society Series B 1995;57:289-300. p
g
4. Fisher, R. A. Frequency distribution of the values of the correlation coefficient in samples from an
indefinitely large population. Biometrika 1915;10:507-521. 3. Miller, R. G. Simultaneous Statistical Inference, Springer, 1981. 1. van den Hoff, M. J., A. F. Moorman, et al. Electroporation in 'intracellular' buffer increases cell survival.
Nucleic Acids Res 1992;20:2902.
2. Hazlehurst, L. A., S. A. Enkemann, et al. Genotypic and phenotypic comparisons of de novo and References 3. Miller, R. G. Simultaneous Statistical Inference, Springer, 1981.
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English
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Superior Vena Cava Occlusion
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Definitions
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cc-by
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Superior Vena Cava Occlusion National Cancer Institute National Cancer Institute Qeios ID: 0D18WW · https://doi.org/10.32388/0D18WW National Cancer Institute. Superior Vena Cava Occlusion. NCI Thesaurus. Code C36065. National Cancer Institute. Superior Vena Cava Occlusion. NCI Thesaurus. Code C36065. Blockage of the lumen of the superior vena cava. Qeios ID: 0D18WW · https://doi.org/10.32388/0D18WW 1/1
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English
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Homochiral Columns Constructed by Chiral Self-Sorting During Supramolecular Helical Organization of Hat-Shaped Molecules
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Journal of the American Chemical Society
| 2,014
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cc-by
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The “hat-shaped” dendronized CTV assembles in bent-branch pine-tree
columns that allow interdigitation of alkyl groups in adjacent columns regardless of their direction. Enantiomerically rich,
racemic, and achiral compositions undergo deracemization in the crystal state by transfer of the transient disc-like conformer of
dendronized CTV from column to column during crown inversion. Solid state NMR experiments identified motional processes
that allow such transfer. This unprecedented supramolecular chiral self-sorting will impact the creation of functions in complex
systems. ABSTRACT: A library of dendronized cyclotriveratrylene
(CTV) crowns substituted with chiral, racemic, or achiral
peripheral alkyl chains, including enantiopure R and S
branched alkyls, “racemic by mixture”, “racemic by synthesis”,
n-octyl, and n-dodecyl groups was synthesized. In solvophobic
solvents and in bulk they self-assemble in helical columns. Their solution and bulk shape-persistent supramolecular
structures were determined by a complementary combination
of circular dichroism (CD) and UV in solution and thin film,
microspot CD in thin film, differential scanning calorimetry
combined with fiber X-ray diffraction, computer simulation, and molecular models. In solution, self-assembly via a cooperative
mechanism generates single-handed columns from enantiopure CTVs and mixtures of right- and left-handed columns from
racemic by mixture, racemic by synthesis, other combinations of R and S, and even from achiral compounds. In bulk state all
supramolecular columns form a 3D hexagonal crystalline phase, Φh
k (P63 symmetry), that can be obtained only from single-
handed columns and a columnar hexagonal 2D liquid crystal, Φh. The highest order Φh
k consists of enantiopure single-handed
columns that are slightly distorted 12-fold triple helices. The “hat-shaped” dendronized CTV assembles in bent-branch pine-tree
columns that allow interdigitation of alkyl groups in adjacent columns regardless of their direction. Enantiomerically rich,
racemic, and achiral compositions undergo deracemization in the crystal state by transfer of the transient disc-like conformer of
dendronized CTV from column to column during crown inversion. Solid state NMR experiments identified motional processes
that allow such transfer. This unprecedented supramolecular chiral self-sorting will impact the creation of functions in complex
systems. heir solution and bulk shape-persistent supramolecular
uctures were determined by a complementary combination
circular dichroism (CD) and UV in solution and thin film,
crospot CD in thin film, differential scanning calorimetry
mbined with fiber X-ray diffraction, computer simulation, and molecular models. In solution, self-assembly via a cooperative
echanism generates single-handed columns from enantiopure CTVs and mixtures of right- and left-handed columns from
cemic by mixture, racemic by synthesis, other combinations of R and S, and even from achiral compounds. In bulk state all
pramolecular columns form a 3D hexagonal crystalline phase, Φh
k (P63 symmetry), that can be obtained only from single-
nded columns and a columnar hexagonal 2D liquid crystal, Φh. The highest order Φh
k consists of enantiopure single-handed
lumns that are slightly distorted 12-fold triple helices. The “hat-shaped” dendronized CTV assembles in bent-branch pine-tree
lumns that allow interdigitation of alkyl groups in adjacent columns regardless of their direction. Enantiomerically rich,
cemic, and achiral compositions undergo deracemization in the crystal state by transfer of the transient disc-like conformer of
ndronized CTV from column to column during crown inversion. Solid state NMR experiments identified motional processes
at allow such transfer. This unprecedented supramolecular chiral self-sorting will impact the creation of functions in complex
stems. ABSTRACT: A library of dendronized cyclotriveratrylene
(CTV) crowns substituted with chiral, racemic, or achiral
peripheral alkyl chains, including enantiopure R and S
branched alkyls, “racemic by mixture”, “racemic by synthesis”,
n-octyl, and n-dodecyl groups was synthesized. In solvophobic
solvents and in bulk they self-assemble in helical columns. Their solution and bulk shape-persistent supramolecular
structures were determined by a complementary combination
of circular dichroism (CD) and UV in solution and thin film,
microspot CD in thin film, differential scanning calorimetry
combined with fiber X-ray diffraction, computer simulation, and molecular models. In solution, self-assembly via a cooperative
mechanism generates single-handed columns from enantiopure CTVs and mixtures of right- and left-handed columns from
racemic by mixture, racemic by synthesis, other combinations of R and S, and even from achiral compounds. In bulk state all
supramolecular columns form a 3D hexagonal crystalline phase, Φh
k (P63 symmetry), that can be obtained only from single-
handed columns and a columnar hexagonal 2D liquid crystal, Φh. The highest order Φh
k consists of enantiopure single-handed
columns that are slightly distorted 12-fold triple helices. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 †Roy & Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323,
United States ‡Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6396, United States
§RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
∥Max-Planck Institute for Polymer Research, Mainz 55128, Germany
⊥Department of Materials Science and Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom
#D
f Ph
Zh
S
T
h U
H
h
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1
Ch ‡Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6396, United States
§RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
∥Max-Planck Institute for Polymer Research, Mainz 55128, Germany p
y
y,
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,
p
,
y
,
§RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
∥Max-Planck Institute for Polymer Research, Mainz 55128, Germany
⊥Department of Materials Science and Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom
#Department of Physics, Zhejiang Sci-Tech University, Hangzhou 310018, China ∥Max-Planck Institute for Polymer Research, Mainz 55128, Germany
⊥Department of Materials Science and Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom
#Department of Physics, Zhejiang Sci-Tech University, Hangzhou 310018, China ⊥Department of Materials Science and Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom
#Department of Physics, Zhejiang Sci-Tech University, Hangzhou 310018, China *
S Supporting Information eratrylene
or achiral
R and S
ynthesis”,
vophobic
columns. molecular
mbination
thin film,
lorimetry
ulation, and molecular models. In solution, self-assembly via a cooperative
enantiopure CTVs and mixtures of right- and left-handed columns from
mbinations of R and S, and even from achiral compounds. In bulk state all
alline phase, Φh
k (P63 symmetry), that can be obtained only from single-
d crystal, Φh. The highest order Φh
k consists of enantiopure single-handed
es. The “hat-shaped” dendronized CTV assembles in bent-branch pine-tree
in adjacent columns regardless of their direction. Enantiomerically rich,
zation in the crystal state by transfer of the transient disc-like conformer of
own inversion. Solid state NMR experiments identified motional processes
olecular chiral self-sorting will impact the creation of functions in complex BSTRACT: A library of dendronized cyclotriveratrylene
CTV) crowns substituted with chiral, racemic, or achiral
ripheral alkyl chains, including enantiopure R and S
anched alkyls, “racemic by mixture”, “racemic by synthesis”,
octyl, and n-dodecyl groups was synthesized. In solvophobic
lvents and in bulk they self-assemble in helical columns. © 2014 American Chemical Society Journal of the American Chemical Society Reduction of the ester to give the benzyl alcohol and
subsequent reaction with thionyl chloride yielded the benzyl
chloride (Scheme SS1, Supporting Information). In the final
step of the synthesis six dendrons were attached to the
demethylated CTV derivative CTV(OH)6 to form the target
dendronized CTV (Scheme SS2, Supporting Information). The
pure enantiomers, (S)-(3,4)dm8*G1-CTV and (R)-(3,4)-
dm8*G1-CTV, were obtained following this synthetic method,
starting from the enantiopure branched bromoalkanes (S)-3,7-
dimethyloctyl bromide and (R)-3,7-dimethyloctyl bromide,
respectively. The compound, named “fully racemized-(3,4)-
dm8*G1-CTV”, “an irreversible racemized CTV”, also referred
in the rest of this report as “racemic by synthesis”, was obtained
by synthesis with the racemic 3,7-dimethyloctyl bromide. This
compound is not a single chemical species but rather a mixture
of stereoisomers. In the first alkylation step of this synthesis
four distinct stereoisomers ((3S,4S), (3S,4R), (3R,4S), and
(3R,4R), respectively) are formed by statistical addition of S or
R alkyl chains at the 3 and 4 positions of the benzyl ring. These
stereoisomers were not separated, and therefore, the benzyl
chloride derivative obtained is still a mixture of four
stereoisomers. Statistical addition of these stereoisomers to
each of the six positions of the CTV core in the final synthetic
step gives rise to an increased number of stereoisomers in the
final product. Thus, the racemic by synthesis-(3,4)dm8*G1-
CTV is a mixture of stereoisomers where molecules have S and
R stereogenic centers in random proportions and positions at
their 12 peripheral alkyl chains. Besides compounds (S)-
(3,4)dm8*G1-CTV, (R)-(3,4)dm8*G1-CTV, and racemic by
synthesis-(3,4)dm8*G1-CTV depicted in Figure 1, additional
samples were prepared by mixing the pure enantiomers (S)-
(3,4)dm8*G1-CTV and (R)-(3,4)dm8*G1-CTV in various Dendronized cyclotriveratrylenes (CTV) provide weakly
interacting and dynamic dendritic crowns that self-assemble
into helical columns and spheres13 resembling the structure of
the cross-section of the helical columns assembled from
dendritic dipeptides.11,12 The flexible dendronized CTV
undergoes crown inversion both in solution and in the bulk
state.13b Therefore, the pine-tree helical columns assembled
from CTV derivatives are expected to provide access to the
mechanisms of formation of chiral supramolecular columns and
their racemization and deracemization. This publication reports
the first study of the mechanism and thermodynamics of self-
assembly in solution and self-organization in the solid state of
enantiomerically pure homochiral, several different mixtures of
homochiral, two different chiral racemic, and achiral CTV
derivatives dendronized with the smallest possible amphiphilic
“minidendron”7m,9b to accomplish fast dynamics. Journal of the American Chemical Society Together with
the first demonstration of deracemization or spontaneous
optical resolution of supramolecular assemblies in the crystal
state this study will demonstrate that dendronized CTV
provides an excellent model for investigation of racemization
and deracemization during supramolecular helical polymer-
ization into complex columnar systems. Journal of the American Chemical Society Journal of the American Chemical Society Article Article Figure 1. Structures of chiral and achiral dendronized cyclo-
triveratrylene (CTV) derivatives. blocks, their supramolecular structures, and their role in the
understanding of fundamental concepts that mediate functions
in complex systems are in demand.10 blocks, their supramolecular structures, and their role in the
understanding of fundamental concepts that mediate functions
in complex systems are in demand.10 p
y
Our laboratory developed a class of dendritic dipeptides that
self-assemble into porous helical transmembrane protein
mimics that are persistent both in solution and in the solid
state.11 Therefore, their self-assembly could be investigated by a
combination of complementary solution and solid state
techniques.1c,12 The role of all stereochemical permutations
of a dendritic dipeptide, including homochiral, heterochiral, and
differentially racemized variants, on the mechanism and
thermodynamics of self-assembly was reported.1c,12 This
study demonstrated that the highest degree of stereochemical
purity, enantiopure homochiral dendritic dipeptides, provides
the most thermodynamically favorable self-assembly process in
solution, corresponding to the greatest degree of order and
crystallization in the solid state.12a Due to the high enthalpy of
supramolecular helical polymerization mediated by a network
of H-bonding and other interactions, these structures, including
those generated with the lowest stereochemical purity, are more
closely related to covalent rather than supramolecular polymers
and subsequently do not undergo deracemization in the solid
state. Therefore, while these experiments demonstrated the
need of biological homochirality1c,12 by showing that
heterochiral and racemic systems provide less perfect
assemblies that would not resemble the homochiral world in
which we inhabit, they did not generate models to investigate
the mechanism of racemization and deracemization. Deracem-
ization experiments, also known as spontaneous optical
resolution or chiral self-sorting, would require a more dynamic
and weakly interacting model that also creates supramolecular
assemblies persistent in solution and in the solid state. Figure 1. Structures of chiral and achiral dendronized cyclo-
triveratrylene (CTV) derivatives. dendron bears two peripheral alkyl chains, which can be either
chiral branched chains ((S)- or (R)-3,7-dimethyloctyl (denoted
dm8*)) or achiral linear chains (denoted 8 for −C8H17 and 12
for −C12H25). The synthesis and characterization of the
compounds from Figure 1 are reported in the Supporting
Information (Schemes SS1 and SS2). Their synthesis is based
on reported procedures.13a,14 Preparation of the (3,4)G1-
CO2CH3 dendron consisted of the alkylation of methyl 3,4-
dihydroxybenzoate using the appropriate bromoalkanes (3,7-
dimethyloctyl bromide, 1-bromooctane, or 1-bromododecane). ■INTRODUCTION involves mechanisms of transfer and amplification of structural
information mediated by a helical arrangement of building
blocks. The complexity of these mechanisms has only begun to
be elucidated.8 Crystallization of supramolecular homochiral
and racemic systems as well as their deracemization entails the
development of methodologies that are available for molecular
but not supramolecular systems.6 Knowledge from these
developments will impact many areas of supramolecular
chemistry and materials including organic electronics,10 bio-
logical mimics, and many others. Therefore, the models
required to study various classes of self-assembling building Most natural compounds and biomacromolecules are homo-
chiral. The origin of biological homochirality has fascinated the
scientific community for centuries,1 and the significance of
homochirality is accepted in biology,1 complex natural
products,2a−c and pharmaceuticals.2d Since the pioneering
experiment of Pasteur,3a the mechanisms of crystallization of
low molar mass racemic organic compounds,3b their deracem-
ization,3,4 how to accomplish it,5 and more recently the
enantioselective synthesis6 have become established fields. However, the generation and mechanisms of action of
molecular and supramolecular chirality in the creation of
supramolecular chiral assemblies are more recent and complex
topics of research.7−9 Creation of supramolecular chirality Received:
April 8, 2014
Published: April 23, 2014 © 2014 American Chemical Society 7169 dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 ■RESULTS AND DISCUSSION Synthesis of Chiral and Achiral Dendronized Cyclo-
triveratrylenes. The structures of the dendronized CTV
derivatives used are shown in Figure 1. All compounds have the
same CTV crown-like core and differ only in their peripheral
alkyl chains. The CTV core is hexasubstituted with first-generation self-
assembling (3,4)G1-CH2 benzyl ether minidendrons. Each 7170 dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society Article Figure 2. DSC traces of (R)- and (S)-(3,4)dm8*G1-CTVs, of their mixtures with different compositions, and of the racemic by synthesis
dendronized CTV recorded with heating and cooling rates of 10 °C/min. The right column shows the heating traces after annealing at the indicated
temperature and time, cooling to −40 °C, and reheating. Phases determined by XRD, transition temperatures, and associated enthalpy changes (in
brackets in kcal/mol) are indicated (Φh
k = columnar hexagonal crystal; Φh = 2D columnar hexagonal liquid crystal; i = isotropic liquid). Figure 2. DSC traces of (R)- and (S)-(3,4)dm8*G1-CTVs, of their mixtures with different compositions, and of the racemic by synthesis
dendronized CTV recorded with heating and cooling rates of 10 °C/min. The right column shows the heating traces after annealing at the indicated
temperature and time, cooling to −40 °C, and reheating. Phases determined by XRD, transition temperatures, and associated enthalpy changes (in
brackets in kcal/mol) are indicated (Φh
k = columnar hexagonal crystal; Φh = 2D columnar hexagonal liquid crystal; i = isotropic liquid). The first three columns from the left part of Figure 2 show
DSC traces collected during first heating, first cooling, and
second heating scans, respectively. The right column provides
heating data recorded after annealing at 60 °C for 2 h and at 40
°C for 24 h followed by cooling to −40 °C. ratios: 1:3, 3:1, and 1:1. The 1:1 mixture, also referred to as
racemic by mixing, differs from the racemic by synthesis or fully
racemized-(3,4)dm8*G1-CTV described above. The racemic
by mixing is a mixture of only two distinct chemical species that
are enantiomers: (S)-(3,4)dm8*G1-CTV (which contains only
S chains) and (R)-(3,4)dm8*G1-CTV (which contains only R
chains). Since racemic by mixing and racemic by synthesis have
very distinct compositions, they are expected to have different
self-assembly behaviors. These differences will help us under-
stand the role of chirality in the helical supramolecular
polymerization of dendronized CTV derivatives. ■RESULTS AND DISCUSSION At 60 °C the crown conformation of
CTV undergoes an umbrella-like inversion (to be discussed in broad peak centered at about 52 °C appeared on the first
heating scan. Nevertheless, the isotropization temperature
remained the same, suggesting that the stability of the 2D Φh
periodic array is not affected by the enantiomeric composition
of the mixture. A “majority-rules” experiment that will be
discussed later demonstrated that in mixtures of 75% (S)/25%
(R) and 75% (R)/25% (S) almost all supramolecular columns
possess the same handedness as the enantiomerically pure
columns. Therefore, the decrease in crystal melting temperature
of the R/S mixtures is attributed to crystals generated from
nonperfect columns with compositions ranging from enantio-
merically pure to enantiomerically rich. Columns with identical
handedness containing both enantiomers cannot establish a
perfectly ordered intercolumnar correlation in Φh
k and,
therefore, form less ordered lower melting crystals dependent
on the defect density in the columns. The stability of the 2D Φh
structure is not affected since it does not depend on the 3D
correlation between columns. The same effect is even more
clear with 50% (S)/50% (R). The melting peak of this
crystalline phase splits into three broader peaks, which implies
formation of crystal domains with different stabilities due to the
inhomogeneous mixing of enantiomers. Interestingly, upon
annealing at 60 °C for 2 h, cooling to −40 °C, and reheating, all
mixtures (75% (S)/25% (R), 25% (S)/75% (R), and 50% (S)/
50% (R)) showed again a single melting temperature that is
identical to that of the pure enantiomers (compare the fourth
and third columns in Figure 2). These results indicate a
deracemization or spontaneous resolution of the columns
containing different enantiomeric excesses that occurred during
annealing at 60 °C in the Φh
k phase for 2 h. This
deracemization makes the columns become enantiomerically
pure in order to enhance their crystal order. The deracemiza-
tion process implies diffusion of molecules between columns at
the annealing temperature. At 60 °C the crown conformation of
CTV undergoes an umbrella-like inversion (to be discussed in This process was studied by solid state NMR and will be
discussed in a different subsection. ■RESULTS AND DISCUSSION Finally, the
achiral derivatives (3,4)8G1-CTV and (3,4)12G1-CTV were
also synthesized following identical procedures to compare
their self-assembly behavior with that of chiral dendronized
CTV. y
g
The pure R or S enantiomers showed identical traces during
first and second heating scans and during first cooling scan with
two phase transitions at 74 or 73 °C and 79 or 78 °C,
respectively. This difference is most probably due to the slightly
lower enantiomeric purity of S. Analysis by X-ray diffraction
(XRD to be discussed later) demonstrated a hexagonal
crystalline phase (Φh
k) at temperatures below 73 °C and a
2D hexagonal liquid crystalline (Φh) phase in the small
temperature region between the crystalline phase and the
isotropic liquid (74 or 73 to 79 or 78 °C). After annealing at 60
°C in the crystal state (selection of this temperature will be
discussed in the NMR section) for 2 h and cooling to −40 °C,
subsequent heating scans of enantiomerically pure S and R
showed the same traces as that obtained during second heating,
except that melting occurred at 74 °C and isotropization at 79
°C for both R and S, indicating that the annealing process did
not further improve the order of the crystalline phase (right
side column in Figure 2). By mixing the two enantiomers in a
ratio of 75% (S)/25% (R) or 75% (R)/25% (S), the melting
temperature of the crystalline phase decreased and a small new Thermal Analysis of Chiral, Racemic, and Achiral
Dendronized CTVs by Differential Scanning Calorim-
etry. Figure 2 shows the differential scanning calorimetry
(DSC) traces of the chiral enantiopure (S)- and (R)-
(3,4)dm8*G1-CTV, of their 75% (S)/25% (R), 25% (S)/
75% (R), and 50% (S)/50% (R) mixtures, and of the fully
racemized or racemic by synthesis CTV samples at heating and
cooling rates of 10 °C/min. The sample with 50% (S)/50% (R)
composition is the racemic by mixing. 7171 dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society Journal of the American Chemical Society Article Figure 3. Sequence of events taking place during the deracemization process between enantiomerically rich columns assembled from the hat-shaped
dendronized CTV (a) via a temporary disc-like conformation moving from column to column during the CTV crown inversion (b, c, d). A movie file
is available in the Supporting Information. Figure 3. ■RESULTS AND DISCUSSION Sequence of events taking place during the deracemization process between enantiomerically rich columns assembled from the hat-shaped
dendronized CTV (a) via a temporary disc-like conformation moving from column to column during the CTV crown inversion (b, c, d). A movie file
is available in the Supporting Information. the NMR section); during its transient disc-like conformation,
the dendronized CTV moves from column to column (Figure
3). A movie that includes the snapshots in Figure 3 is available
in the Supporting Information. broad peak centered at about 52 °C appeared on the first
heating scan. Nevertheless, the isotropization temperature
remained the same, suggesting that the stability of the 2D Φh
periodic array is not affected by the enantiomeric composition
of the mixture. A “majority-rules” experiment that will be
discussed later demonstrated that in mixtures of 75% (S)/25%
(R) and 75% (R)/25% (S) almost all supramolecular columns
possess the same handedness as the enantiomerically pure
columns. Therefore, the decrease in crystal melting temperature
of the R/S mixtures is attributed to crystals generated from
nonperfect columns with compositions ranging from enantio-
merically pure to enantiomerically rich. Columns with identical
handedness containing both enantiomers cannot establish a
perfectly ordered intercolumnar correlation in Φh
k and,
therefore, form less ordered lower melting crystals dependent
on the defect density in the columns. The stability of the 2D Φh
structure is not affected since it does not depend on the 3D
correlation between columns. The same effect is even more
clear with 50% (S)/50% (R). The melting peak of this
crystalline phase splits into three broader peaks, which implies
formation of crystal domains with different stabilities due to the
inhomogeneous mixing of enantiomers. Interestingly, upon
annealing at 60 °C for 2 h, cooling to −40 °C, and reheating, all
mixtures (75% (S)/25% (R), 25% (S)/75% (R), and 50% (S)/
50% (R)) showed again a single melting temperature that is
identical to that of the pure enantiomers (compare the fourth
and third columns in Figure 2). These results indicate a
deracemization or spontaneous resolution of the columns
containing different enantiomeric excesses that occurred during
annealing at 60 °C in the Φh
k phase for 2 h. This
deracemization makes the columns become enantiomerically
pure in order to enhance their crystal order. The deracemiza-
tion process implies diffusion of molecules between columns at
the annealing temperature. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 ■RESULTS AND DISCUSSION After annealing at 40 °C
for 24 h, only a fraction of crystals increased their melting
temperature to 48 °C, suggesting that after an extended period
of annealing in the crystalline phase the racemic by synthesis
sample cannot improve the crystal order to the same extent as
that observed in pure enantiomers. This indicates that it is not
possible to achieve perfect 3D column to column correlation in
Φh
k if complete deracemization of CTV enantiomers is not
accessible at the molecular level. Even in this case, all columns
from the crystal domain must have overall the same handedness
with reverse rotation sense molecules as defects (see below the
discussion of XRD and CD). In summary, the difference in
phase stability and crystallization behavior is greatly affected by
the enantiomeric composition within columns. The pure R or S
enantiomers tend to stack into columns with selected
handedness, and columns with the same handedness and
molecular chirality can pack with each other three dimension-
ally with long-range order to form stable Φh
k crystals possessing
a high melting temperature. The Φh
k phase consisting of
monodomains of left- or right-handed supramolecular columns
formed from various ratios of CTV enantiomers resemble low
molar mass conglomerates, albeit produced according to more
complex principles (compare Scheme 1 and Scheme SS3,
Supporting Information).3b pp
g
The c axis is along the column axis which is also parallel to
the fiber direction. All samples show sharp equatorial (hk0)
reflections corresponding to a well-ordered hexagonal array. The numerous strong and sharp (hkl) reflections observed for
pure enantiomers (Figures 5m, 7a, and 7d) indicate a long-
range ordered crystal structure (Φh
k) with highly correlated
molecular positions between neighboring columns. The four
layer lines perpendicular to the fiber axis would suggest a 41
helical columnar structure. As the molecule itself has 3-fold C3v
symmetry, it would be more appropriate to view the column as
a 121 triple helix or a 123 helix. Note that a +90° rotation
between successive C3v molecules, implied by a right 4-fold
helix, is equivalent to a −30° rotation of a left 12-fold helix. However, we note that the above refers only to isolated
columns. Once put in a periodic hexagonal 3D lattice, the 41 or
123 symmetries become only approximations, as they are
incompatible with any hexagonal space group. ■RESULTS AND DISCUSSION Annealing in the 2D Φh
phase does not induce deracemization since this spontaneous
optical resolution or chiral self-sorting is driven by establish-
ment of higher 3D intercolumnar order in the Φh
k structure,
which demands all columns to have the same handedness in a
crystalline domain (Figure 4 and to be detailed in a later
subsection). In the racemic by synthesis dendronized CTV R
and S configurations are distributed randomly among the 12
side chains. The DSC curve of this sample shows only one Figure 4. Schematic representation of columnar crystalline hexagonal
lattice (Φh
k) with the unit cell (red box) indicated in (a) top view and
(b) tilt view. Each unit cell contains one single column which implies
single-helical handedness for all columns in the crystal. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185
2 Figure 4. Schematic representation of columnar crystalline hexagonal
lattice (Φh
k) with the unit cell (red box) indicated in (a) top view and
(b) tilt view. Each unit cell contains one single column which implies
single-helical handedness for all columns in the crystal. 7172 Journal of the American Chemical Society Article Scheme 1. Crystallization of a Racemic Mixture of Self-Assembling R and S Building Blocks Forming Columnar Assemb
aTwo sequences of R and S enantiomers are shown for conglomerates of enantiomerically rich columns (bottom and middle left side). Scheme 1. Crystallization of a Racemic Mixture of Self-Assembling R and S Building Blocks Forming Columnar Assembliesa on of a Racemic Mixture of Self-Assembling R and S Building Blocks Forming Columnar Assembliesa R and S enantiomers are shown for conglomerates of enantiomerically rich columns (bottom and middle left side). aTwo sequences of R and S enantiomers are shown for conglomerates of enantiomerically rich columns (bottom and pounds. The fiber diffraction pattern of the R enantiomer of
(3,4)dm8*G1-CTV is shown in Figure 5m, and that of its
achiral or nonchiral analogue with linear pendant chains,
(3,4)8G1-CTV, is shown in Figure 6. Both are in the crystalline
Φh
k phase. The 3D order is evident in both patterns by the
presence of discrete Bragg reflections on a number of layer
lines. The observed indexed Bragg spacings are listed in Table
ST2, Supporting Information. broad crystal melting peak at lower temperature (37 °C) upon
second heating. This transition corresponds to the melting of
low-ordered crystals created by the poor packing of columns
generated from the two enantiomers. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society Article Figure 5. Schematic models of (a, d, g) two successive CTV molecules within a column, (b, e, h) packing of two dimers viewed along the c axis, and
(c, f, (i) supramolecular column with approximate triple 121 helix conformation and P63 crystal symmetry for the three models with different side
chain orientations. Comparison of the simulated fiber XRD patterns from model 1 (j), model 2 (k), and model 3 hat-shaped (l) with the
experimental pattern (m) of the 3D Φh
k crystalline phase of the R enantiomer of (3,4)dm8*G1-CTV. The fourth layer line intensity scans obtained
in the red box region are shown at the bottom. Journal of the American Chemical Society
Article Figure 5. Schematic models of (a, d, g) two successive CTV molecules within a column, (b, e, h) packing of two dimers viewed along the c axis, and
(c, f, (i) supramolecular column with approximate triple 121 helix conformation and P63 crystal symmetry for the three models with different side
chain orientations. Comparison of the simulated fiber XRD patterns from model 1 (j), model 2 (k), and model 3 hat-shaped (l) with the
experimental pattern (m) of the 3D Φh
k crystalline phase of the R enantiomer of (3,4)dm8*G1-CTV. The fourth layer line intensity scans obtained
in the red box region are shown at the bottom. Figure 6. Comparison of the experimental and simulated fiber XRD
patterns of (3,4)8G1-CTV. Column model used in the simulation is
shown on the right. axis normal to the 3-fold axis, only the P63 space group (space
group no. 173) remains. The possibility that these additional
lattice symmetry elements may be present due to random
inversions of the “hat-shaped dendronized CTV” is not
excluded, since “hat inversions” do not racemize the system,
as shown by circular dichroism (see below). Therefore, the
P6322 space group is less probable. p
g
p
p
Since the Φh
k unit cell contains a single column the above
implies that all 4 molecules in the cell and all columns in the
crystal domain possess the same helical sense (Figure 4).15 y
p
( g
)
As already noted, strict 123 or 41 screw axis symmetry is
incompatible with hexagonal crystal symmetry. The true
crystallographic symmetry is 63, or triple 21, and takes account
of the column environment, not just the column itself. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 ■RESULTS AND DISCUSSION The hexagonal unit cell parameters for the two enantiomers
of (3,4)dm8*G1-CTV are a = 34.3 Å (the “column diameter”)
and c = 18.5 Å. The distance between two molecules along the
column axis is 4.6 Å (the d spacing of the (004) reflection), and
the pitch (full turn) of the 12-fold helix is 55.5 Å. That there are pp
g
X-ray Fiber Diffraction and Molecular Models of the
Supramolecular Columns from Enantiopure (3,4)-
dm8*G1-CTV and from Achiral (3,4)8G1-CTV Com- 7173 Journal of the American Chemical Society Journal of the American Chemical Society In
both models combinations of up and down columns are allowed by the hat-shaped dendronized CTV (see also Figure SF10b, Supporting
Information). Journal of the American Chemical Society
Article Figure 7. Comparison of the experimental and simulated fiber XRD patterns of the Φh
k 3D crystalline phase of the (a) R and (d) S enantiomers. Models of the supramolecular columns seen from the top and side views constructed by the (b) R and (e) S enantiomers. The helical sense was
determined by CD-UV experiments. Chiral centers are decorated as dark/light blue for the R enantiomer and dark/light green for the S enantiomer
for easy visualization of the helical structure. Crystal packing models for top and side views of the (c) R and (f) S enantiomers in the Φh
k lattice. In
both models combinations of up and down columns are allowed by the hat-shaped dendronized CTV (see also Figure SF10b, Supporting
Information). crystal packing exerts on the columnar helix. The resulting
supramolecular column is depicted in Figure 5c, 5f, and 5i as
discussed below. affected mostly by the orientation of the dendron and its alkyl
groups (bottom plots of Figure 5j, 5k, and 5l). When we
compare the simulated and experimental fourth layer line scans,
it becomes clear that model 3 based on the hat-shaped
dendronized CTV best agrees with the data. In model 3 the
chains are straight and tilted near the junctions with the
benzene rings but become horizontal toward the chain ends. This suggests that as moving toward the chain ends in the chiral
compounds, the branching and increased conformational
disorder (likelihood of gauche bonds) increase the effective
chain cross-section and lead to their horizontal conformation. Model 2 from Figure 5f requires an up−down alternation
between the pine-tree columns (antiparallel) in the Φh
k phase
to ensure effective packing between the alkyl groups. Journal of the American Chemical Society Thus,
the asymmetric unit is in fact a dimer (Figure 5a, 5d, and 5g),
the unit cell containing two dimers related to each other by a 21
operation. The two molecules in the dimer are not mutually
related by crystal symmetry but are similar and related
approximately by the 41 or 123 symmetry, i.e., by a 90° or
30° rotation. A second dimer is seated on top of the first one,
rotated by 180° (21) or 60° (63) relative to the first dimer, and
moved up by one-half of the Φh
k unit cell along the c axis to
generate a continuous helix with intermolecular rotation of 30°
(Figure 5b, 5e, and 5h). The absence of the 002 reflection is
thus not a systematic absence, as the space group allows it. This
is a common situation, and 00l reflections forbidden by the
apparent helical symmetry of the column are often observ-
ed.10c,d Their intensity reflects the degree of distortion that Figure 6. Comparison of the experimental and simulated fiber XRD
patterns of (3,4)8G1-CTV. Column model used in the simulation is
shown on the right. 4 molecules per unit cell has been confirmed by the comparison
of unit cell volume and experimental density (1.04 g/cm3)
(Table ST3, Supporting Information). There is only one
column per unit cell. The columns are thus assembled by the
stacking of CTV cores with one molecule per column stratum
of 4.6 Å. With the absence of meridional 00l reflections with l =
odd and with no other systematic extinctions there are three
possible space groups: P63, P63/m, or P6322. However, since
the molecules do not possess either a mirror plane or a 2-fold 7174 Journal of the American Chemical Society Article Figure 7. Comparison of the experimental and simulated fiber XRD patterns of the Φh
k 3D crystalline phase of the (a) R and (d) S enantiomers. Models of the supramolecular columns seen from the top and side views constructed by the (b) R and (e) S enantiomers. The helical sense was
determined by CD-UV experiments. Chiral centers are decorated as dark/light blue for the R enantiomer and dark/light green for the S enantiomer
for easy visualization of the helical structure. Crystal packing models for top and side views of the (c) R and (f) S enantiomers in the Φh
k lattice. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society (f−i) Patterns collected at 20 °C after annealing at
60 °C for 2 h and cooling to 20 °C with a rate of 10 °C/min, and (j) annealing at 40 °C for 24 h and cooling to 20 °C with a rate of 10 °C/min. Fiber axis, layer lines (L), temperature, phase, and lattice parameters are indicated. Figure SF10, Supporting Information). Conversely, for the
columns with linear achiral side groups the chains are more
tilted (closer to model 2 in Figure 5k) as their effective cross-
section is narrower. A diffraction feature associated with the
chain tilt can be readily identified in both the simulated and the
experimental patterns of (3,4)8G1-CTV (red oval in Figure 6). However, the weaker meridian reflection in the simulated
pattern (white oval) implies that toward the chain ends the
linear alkyl chains curve toward a horizontal orientation,
although not as much as in the case of the branched chiral
chains. This behavior is understandable as the probability of the
gauche conformation increases toward the chain end in both
cases, but the extra space required by the branch is not required
in (3,4)8G1-CTV. and disregard the up and down arrangement of adjacent
columns. In contrast, the structure generated by mixing chiral and
linear CTV compounds loses the tilted chain feature and
displays no crystal order (Figure SF2, Supporting Information),
indicating that mixing inhibits the preferred alkyl chain
conformation for each compound and thus makes it difficult
to establish crystal order between neighboring columns (Figure
4). The supramolecular columnar structures, crystal models, and
comparison between simulated and experimental fiber XRD
patterns of the Φh
k structure for the two enantiomers are
shown in Figure 7. The simulated diffraction patterns based on
the molecular models constructed with the cell symmetry
discussed above are shown in Figure 7a and 7d for the R and S
enantiomers, respectively. The helical sense of the R- and S-
based models was determined by CD-UV experiments to be
discussed in a different subsection. Very good agreement
between the experimental and the simulated patterns is
observed. In the columnar model shown in Figure 7b and 7e
the left side shows the hat-shaped dendronized CTV single
molecule and the columnar model with the chiral centers
highlighted. The 121 helical structure and the relative positions
of the chiral center in the right- or left-handed columns can be
easily identified. Journal of the American Chemical Society This
arrangement would require two columns per unit cell and thus
reduce the hexagonal symmetry of the 3D Φh
k periodic array to
orthorhombic or monoclinic.10b−d This arrangement is not
demanded by the bent-branch pine-tree columns generated
from the hat-shaped dendronized CTV (model 3 in Figure 5i)
since the packing of the alkyl group in adjacent columns does
not depend on the up or down orientation of the columns (for
comparison of the two column packing models 2 and 3 see We proceeded to build detailed molecular models and fit the
experimental diffraction pattern to those simulated from the
models. Figure 5 outlines possible molecular models of the
supramolecular columns forming a Φh
k periodic array. Simulations of their fiber XRD patterns and comparison with
the experimental data are also shown. Three models with
identical helical conformation of the CTV columnar core but
with different side chain conformations/orientations were
considered. In model 1, the benzyl ether and alkyl chains are
perpendicular to the column, whereas they are all tilted in
model 2. In model 3, the benzyl ether is tilted and the alkyl
chains start tilting near the junction point of the dendron but
ultimately become perpendicular to the long axis of the column. This arrangement provides an unprecedented “hat-shaped”
dendronized CTV conformation. Simulated fiber XRD patterns
obtained from Φh
k arrays generated from each model are shown
in Figure 5j, 5k, and 5l, respectively. The most significant differences between these patterns are
the intensity distributions along the L = 4 layer line, which are 7175 Journal of the American Chemical Society Article Figure 8. Wide angle X-ray diffraction patterns collected from the oriented fibers of chiral CTVs with indicated composition. (a−e) Patterns
collected at 20 °C after first cooling from 80 to 20 °C with a rate of 10 °C/min without annealing. (f−i) Patterns collected at 20 °C after annealing at
60 °C for 2 h and cooling to 20 °C with a rate of 10 °C/min, and (j) annealing at 40 °C for 24 h and cooling to 20 °C with a rate of 10 °C/min. Fiber axis, layer lines (L), temperature, phase, and lattice parameters are indicated. Figure 8. Wide angle X-ray diffraction patterns collected from the oriented fibers of chiral CTVs with indicated composition. (a−e) Patterns
collected at 20 °C after first cooling from 80 to 20 °C with a rate of 10 °C/min without annealing. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society Article the as-prepared sample, and only a few off-meridional
reflections were observed, suggesting that a large portion of
columns is constructed with random mixtures of enantiomers
(Scheme 1), further hindering correlations between the
neighboring columns of the Φh
k phase. The weaker odd layer
lines in the pattern also suggest that the columns may undergo
very frequent helix reversals as defects in the homochiral Φh
k
crystal domain. Comparison of Chiral Assemblies of Enantiopure R, S,
and Various Enantiomeric Ratios Including “Racemic by
Mixture” and Racemic by Synthesis by Fiber XRD. The
top row in Figure 8 shows the XRD patterns collected at 20 °C
from the Φh
k phase obtained from pure CTV enantiomers,
various mixtures of enantiomers, and racemic by synthesis CTV
collected from oriented fibers after first cooling from 80 to 20
°C. When two enantiomers were mixed with a 75% (S)/25%
(R) or 75% (R)/25% (S) ratio (Figure 8c), the degree of
intracolumnar order and column−column correlation de-
creased. This is seen from the general broadening of the
Bragg reflections but most conspicuously from the weakening
of the odd layer lines (L = 1, 3). One should appreciate that
18.5 Å (4 × 4.6) periodicity (first layer line) comes from the 4
successive molecules twisting in the same direction by 90°. If
that was not the case and the rotation was alternatively by +90°
and −90°, the period would have been only 9.2 Å (2nd layer
line) and the odd layer lines would have not existed. k Even less crystal order was observed in the racemic by
synthesis 3D columnar periodic array (Figure 8e) where
columns are made of molecules with totally random side chain
chirality. The low-order and inhomogeneous crystal structure
gives lower and less defined melting temperatures (Figure 2). Nevertheless, all samples exhibit a sharp and strong 4.6 Å
meridian reflection that indicates a well-defined CTV to CTV
distance of 4.6 Å. This is not surprising because this is the one
feature that is totally independent of the stereochemistry from
the side groups of the dendronized CTV. In other words, the
crown stacking periodicity is not influenced by their side chain
chirality. Journal of the American Chemical Society A simulation of the XRD pattern of the Φh
k phase generated
from nonhelical columns (Figure 9) demonstrated the absence y
The bottom row in Figure 8 shows the XRD patterns
obtained for each top sample after annealing at 60 °C for 2 h
(compare Figure 8a−d with 8f−i) or annealing at 40 °C for 24
h (Figure 8j) where fast motion was observed by solid state
NMR (to be discussed in a later section). No marked
improvement is seen in the crystalline order of the pure
enantiomers (Figure 8f and 8g), as the order had already been
high before annealing. It is very telling, however, that the crystal
order of the mixtures of enantiomers was improved greatly
(Figure 8h and 8i). In particular, the pronounced strengthening
of the L = 1 and 3 layer line reflections after annealing
(compare Figure 8a−d with 8f−i) shows that even in random
enantiomeric mixtures, including the 50% (S)/50% (R) blend,
coherent crystal domains of considerable size are formed
consisting of single-handed helical columns since the XRD
patterns became similar to those recorded from enantiomeri-
cally pure columnar structures. Figure 9. Simulated fiber crystal XRD pattern and the corresponding
schematic columnar structure for (a) triple 121 helical structure and
(b) nonhelical structure generated by alternating +30°, −30° rotation
of molecules along column. Loss of odd layer lines is evident in the
nonhelical structure. y p
Therefore, these results demonstrate deracemization of
enantiomers and phase separation of helical columns with
monodomains of left- and right-handed columns forming Φh
k
during the annealing process. However, only a slight improve-
ment of crystal order was identified for the racemic by synthesis
compound even after annealing for 24 h (Figure 8) (since in
this case the random distribution of R and S chains within
monomers cannot drive a complete phase separation of
enantiomerically pure columns). Similarly, the annealing
treatment does not improve the Φh
k order for columnar arrays
generated from achiral compounds with linear chains or
mixtures of chiral and achiral molecules (Figure SF3,
Supporting Information). It is interesting to note that, similarly,
it has been reported that enantiopure helicene molecules also
form large single-handed 3D hexagonal arrays. In their racemic
mixture the two enantiomers form their separate left- and right-
handed helical columns, but in that case both column types
cocrystallize forming a regular alternating array, in this case
monoclinic.15 Figure 9. Journal of the American Chemical Society The right side shows the columnar model with
the potential surface. For clarity, the alkyl chain segments after
the chiral center were cut off. The chiral centers, marked with
dark and light blue for R enantiomers and dark and light green
for S enantiomers, show the triple helix structures with opposite
handedness. Tilted and side views of a model containing 7
enantiomerically pure columns are shown in Figure 7c and 7f. A
hexagonal unit cell is labeled by the white boxes. Only one
column can be accommodated in the unit cell, indicating that
all columns in the crystal are required to possess the same
handedness (see Φh
k unit cell in Figures 4 and 7c). The high
degree of intercolumnar correlation of molecular positions can
be identified from the side view of the unit cell models that
result in a long-range ordered 3D periodic array. Evidence for increased gauche conformation toward chain
ends in n-alkanes is well documented in the literature16 and
confirmed by 13C solid state NMR measurements for (3,4)8G1-
CTV (Figure SF9, Supporting Information). The (3,4)8G1-
CTV with linear side chains showed blurred layer lines in the
XRD pattern, indicating a helical structure with poor long-range
crystal order despite a well-defined local packing observed in
solid state NMR measurements (Figure SF9, Supporting
Information). The first and second layer lines in the simulated
pattern have their maxima closer to meridian than those in the
experimental XRD, indicating that the actual helical structure is
narrower than in the molecular model. This is most likely
because the disordered outer parts of the alkyl chains
contribute only to isotropic diffuse scattering and, therefore,
give a narrower helix and a wider cross. A similar situation is
seen in the diffraction pattern of the DNA helix where the
cross-pattern is narrower in the simulated pattern than in the
experimental one.17 It may be speculated that the reason for
lower intercolumnar order in the linear-chain (3,4)8G1-CTV
compound compared to that in the branched (3,4)dm8*G1-
CTV is the fact that the tilted chains of the former are more
difficult to interdigitate than the horizontal chains of the latter
(Figure SF10, Supporting Information). Effective interdigita-
tion is believed to promote intercolumnar positional correlation dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 7176 Journal of the American Chemical Society dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society This indicates that columnar
aggregates are still present at this temperature. if the supramolecular columns are to reorganize, as discussed in
the earlier DSC and XRD analysis sections. The dynamic
processes can be most easily studied in the racemic by synthesis
CTV sample because of the broad LC phase it exhibits (Figure
2). Moreover, the wealth of information that exists about
molecular motions in columnar discotic LCs19 can be employed
in our analysis. The most prominent molecular motions in 2D
columnar structures are rotations about the column axis. A
recent study combining XRD, dielectric spectroscopy, and solid
state NMR is available.20 Moreover, complex dynamic
processes have been identified, where moieties forming
columnar structures leave the column, flip over, and return to
the columns again.10b Early work on translational diffusion of
disc-like molecules revealed that diffusion between the columns
is much faster than motion along the column.21 This means
that in columns assembled from disc-like molecules rotation of
discs that are most easy to detect by solid state NMR are
intimately linked to processes that allow reorganization of the
columnar structures. Remarkably, a signal due to an aromatic group at about 117
ppm “reappears” at higher temperatures (80−90 °C). It is
assigned to the 6 chemically equivalent proton-carrying
aromatic carbon sites in the crown. The signal of the CH2
groups of the CTV crown at 33 ppm, however, is completely
gone. This indicates that the crown fluctuates between its “up”
and “down” conformations. These conformational changes
cause the 13C−1H dipole−dipole coupling to fluctuate strongly
for all positions, except for these proton-carrying aromatic
carbons, for which the C−H bond directions are close to the
plane of the fluctuating crown. Thus, on average, the crown
behaves like a disc at 50 °C for the racemic by synthesis (Figure
10a) and at 60 °C for the enantiopure (Figure 10b). As stated
above, the prominent motions in LCs self-organized from disc-
like molecules are the axial rotation about the column axis and
the translation between columns. Therefore, we ascribe the
pronounced dynamics detected in the CTVs by NMR to
fluctuations in the columns and exchange between columns,
which offers a mechanism for the deracemization observed in
those range of temperatures both by DSC (Figure 2) and XRD
(Figure 8) experiments. Journal of the American Chemical Society Simulated fiber crystal XRD pattern and the corresponding
schematic columnar structure for (a) triple 121 helical structure and
(b) nonhelical structure generated by alternating +30°, −30° rotation
of molecules along column. Loss of odd layer lines is evident in the
nonhelical structure. of Bragg reflections at L = 1 and 3. Similar disappearance of
odd layer lines is expected if the sequence of positive and
negative molecular rotations was random. It is thus under-
standable that a mixture with a considerable amount of
opposite enantiomer disrupts the sense of intermolecular twist
and thus weakens the odd layer lines disproportionally. These
results are consistent with the DSC analysis, which suggests
that less than 100% enantiomeric purity results in non-
enantiopure columns or even in columns with opposite
handedness that are incompatible with the Φh
k unit cell
requirement of single handedness column, leading to poor
intercolumnar correlations. With a 50% (R)/50% (S) mixture
(Figure 8d) the overall 3D crystal order is further reduced in Analysis of the Molecular Dynamics of the Supra-
molecular Columns Produced from Racemic by Syn-
thesis and Enantiopure S by Variable-Temperature
Solid State
13C NMR Experiments. Solid state NMR
experiments can provide unique information about the time
scale and, due to its site selectivity, the mechanism of molecular
motions of the building blocks.18 Such motions must take place 7177 dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society Journal of the American Chemical Society Article Figure 10. Temperature-dependent 13C CP-MAS NMR spectra of (a) racemic by synthesis dendronized CTV and (b) (S)-(3,4)dm8*G1-CTV,
recorded at 700 MHz 1H Larmor and 25 kHz MAS spinning frequency. Different spectral regions are plotted on different intensity scales, as
indicated. y Figure 10. Temperature-dependent 13C CP-MAS NMR spectra of (a) racemic by synthesis dendronized CTV and (b) (S)-(3,4)dm8*G1-CTV,
recorded at 700 MHz 1H Larmor and 25 kHz MAS spinning frequency. Different spectral regions are plotted on different intensity scales, as
indicated. the 100 kHz to MHz range. At even higher temperatures, these
signals become weaker as the increasing mobility averages the
dipole−dipole coupling to the carbons, which are needed to
generate signals via CP. Note that at 90 °C, i.e., in the molten
state, small signals of the aliphatic carbons as well as those of
aromatics are still detected. Journal of the American Chemical Society It should be noted that the time scales
of the local motions detected here are in the range of tens of
kHz, whereas deracemization occurs on a time scale of hours. This indicates that the building blocks forming the columnar
structures need a large number of motional steps before
equilibrium is reached. This is similar to the transition from
disordered columns of dendronized perylene bisimides to their
thermodynamically controlled state.10b columnar structures. Indeed, well-established 13C cross-polarization (CP) MAS
NMR data already provide a wealth of information. Spectra are
well resolved in well-ordered crystalline states but also in highly
mobile LC systems. The signals severely lose intensity for
moieties involved in motions on the tens of kHz time scale due
to interference effects of the fluctuating dipole−dipole
couplings with the radiofrequency pulses and the MAS rotation
frequency, which can be used to assign molecular motions to
specific sites in complex systems.22 The signals eventually
vanish for rapid isotropic motions in the isotropic liquid.18
NMR spectra of the racemic by synthesis dendronized CTV
sample (Figure 10a) and of the enantiopure sample (Figure
10b) show well-resolved spectra in the solid state at 40 °C,
where all aromatic sites in the CTV unit are resolved. As
indicated, they are grouped as quaternary carbons (130−150
ppm) and aromatic CH groups (110−120 ppm). In the Φh LC
phase at 50 °C (Figure 10a), the aromatic signals broaden
(indicating kHz motions) whereas the aliphatic side groups
become better resolved, indicating rapid anisotropic motions in Indeed, well-established 13C cross-polarization (CP) MAS
NMR data already provide a wealth of information. Spectra are
well resolved in well-ordered crystalline states but also in highly
mobile LC systems. The signals severely lose intensity for
moieties involved in motions on the tens of kHz time scale due
to interference effects of the fluctuating dipole−dipole
couplings with the radiofrequency pulses and the MAS rotation
frequency, which can be used to assign molecular motions to
specific sites in complex systems.22 The signals eventually
vanish for rapid isotropic motions in the isotropic liquid.18 NMR spectra of the racemic by synthesis dendronized CTV
sample (Figure 10a) and of the enantiopure sample (Figure
10b) show well-resolved spectra in the solid state at 40 °C,
where all aromatic sites in the CTV unit are resolved. As
indicated, they are grouped as quaternary carbons (130−150
ppm) and aromatic CH groups (110−120 ppm). dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society Dodecane was chosen as a solvent for
this experiment because it promotes helical self-assembly of the
dendronized CTV.13a,14 At high temperatures the dendronized
CTVs are molecularly dissolved, and no ellipticity is observed
in the CD spectrum since the chiral centers are located in the
peripheral alkyl chains which do not absorb in the UV region. However, upon cooling to lower temperatures, helical supra-
molecular polymerization occurs and chirality is transferred
from the branched side chains to the central aromatic core of
the molecular and supramolecular structure. Growth of the self-
assembled columns upon cooling is reflected by the emergence
of Cotton effects with increasing ellipticity in the CD spectra. The stereochemistry of the peripheral alkyl chains dictates the
helical sense of the columns, as evidenced by the mirror image
CD spectra obtained for the S and R enantiomers of
(3,4)dm8*G1-CTV (Figure 11a). While the helix handedness
of the (S)-(3,4)dm8*G1-CTV self-assembly is clearly the
opposite of the handedness of the (R)-(3,4)dm8*G1-CTV self-
assembled helices, their absolute helical sense is more difficult
to determine. Several contributions are overlapping in the CD
spectra. However, the first coupletwith a peak at 193 nm and
a trough at 206 nm for the S enantiomeris a negative exciton
coupling that corresponds to the main absorption band in the
UV spectrum. According to the exciton coupling theory, this
negative exciton coupling suggests that the S enantiomer of
(3,4)dm8*G1-CTV forms left-handed helical columns.23 CD spectra of the enantiomerically pure (3,4)dm8*G1-CTV
were also recorded in the solid state. Thin films were cast on
quartz plates by spin coating from 2% w/v chloroform solutions
of the dendronized CTV. CD spectra in thin films are nearly
identical to the spectra in solution (Figure 11b). This
equivalence of the spectra in thin film and in solution confirms
that very similar and shape-persistent self-assembled structures
form both in the Φh
k crystal state and in solution. Since CD
spectroscopy is very sensitive to conformational changes, the
high similarity of the spectra confirms that the dendronized
CTV molecules adopt comparable conformations in their self-
assembled state in the bulk state and in solution. The sign of
the Cotton effects is also identical in thin film and in solution
for a given enantiomer, which demonstrates that the helices
have the same handedness in the solid state and in solution. Journal of the American Chemical Society In the Φh LC
phase at 50 °C (Figure 10a), the aromatic signals broaden
(indicating kHz motions) whereas the aliphatic side groups
become better resolved, indicating rapid anisotropic motions in y
y
Analysis of the Helical Supramolecular Polymer-
ization by Circular Dichroism (CD) and UV Absorption
Experiments in Solution and in Thin Film. CD and UV y
y
Analysis of the Helical Supramolecular Polymer-
ization by Circular Dichroism (CD) and UV Absorption
Experiments in Solution and in Thin Film. CD and UV 7178 Journal of the American Chemical Society Article Figure 11. (a) CD and UV spectra of (S)- and (R)-(3,4)dm8*G1-CTV upon cooling from 25 to 0 °C (4.0 × 10−5 M in dodecane). S enantiomer is
in red; R enantiomer is in blue. (b) Thin film CD spectra of S (red bold line) and R (blue bold line) enantiomer at 25 °C. Solution spectra (0 °C)
are also given for comparison (dashed lines, 4.0 × 10−5 M dodecane solutions multiplied by a factor of 2.5). (c) Degree of aggregation (from UV
absorption at 206 nm) collected upon cooling of n-butanol solutions from 50 to −10 °C (total concentration of dendrimers: 4.0 × 10−5 M for the S,
R, and racemic by synthesis sample; 8.0 × 10−5 M for the S/R 50:50 sample), and fitting with the cooperative elongation model (solid lines). (d, e)
Majority-rules experiment. (d) CD spectra collected from mixtures of two enantiomers at 0 °C in dodecane. (e) Net helicity dependence of the
enantiomeric excess (from the ellipticity at 289 nm). Figure 11. (a) CD and UV spectra of (S)- and (R)-(3,4)dm8*G1-CTV upon cooling from 25 to 0 °C (4.0 × 10−5 M in dodecane). S enantiomer is
in red; R enantiomer is in blue. (b) Thin film CD spectra of S (red bold line) and R (blue bold line) enantiomer at 25 °C. Solution spectra (0 °C)
are also given for comparison (dashed lines, 4.0 × 10−5 M dodecane solutions multiplied by a factor of 2.5). (c) Degree of aggregation (from UV
absorption at 206 nm) collected upon cooling of n-butanol solutions from 50 to −10 °C (total concentration of dendrimers: 4.0 × 10−5 M for the S,
R, and racemic by synthesis sample; 8.0 × 10−5 M for the S/R 50:50 sample), and fitting with the cooperative elongation model (solid lines). (d, e)
Majority-rules experiment. Journal of the American Chemical Society (d) CD spectra collected from mixtures of two enantiomers at 0 °C in dodecane. (e) Net helicity dependence of the
enantiomeric excess (from the ellipticity at 289 nm). Conversely, the self-assembled columns formed by the R
enantiomer can be assigned as right-handed helical columns. spectra of the pure enantiomers of (3,4)dm8*G1-CTV in 4.0 ×
10−5 M dodecane solution upon cooling from 25 to 0 °C are
depicted in Figure 11a. Helical supramolecular polymerization
in this solvent is evidenced by the emergence of a bisignated
CD signal upon cooling. Dodecane was chosen as a solvent for
this experiment because it promotes helical self-assembly of the
dendronized CTV.13a,14 At high temperatures the dendronized
CTVs are molecularly dissolved, and no ellipticity is observed
in the CD spectrum since the chiral centers are located in the
peripheral alkyl chains which do not absorb in the UV region. However, upon cooling to lower temperatures, helical supra-
molecular polymerization occurs and chirality is transferred
from the branched side chains to the central aromatic core of
the molecular and supramolecular structure. Growth of the self-
assembled columns upon cooling is reflected by the emergence
of Cotton effects with increasing ellipticity in the CD spectra. The stereochemistry of the peripheral alkyl chains dictates the
helical sense of the columns, as evidenced by the mirror image
CD spectra obtained for the S and R enantiomers of
(3,4)dm8*G1-CTV (Figure 11a). While the helix handedness
of the (S)-(3,4)dm8*G1-CTV self-assembly is clearly the
opposite of the handedness of the (R)-(3,4)dm8*G1-CTV self-
assembled helices, their absolute helical sense is more difficult
to determine. Several contributions are overlapping in the CD
spectra. However, the first coupletwith a peak at 193 nm and
a trough at 206 nm for the S enantiomeris a negative exciton
coupling that corresponds to the main absorption band in the
UV spectrum. According to the exciton coupling theory, this
negative exciton coupling suggests that the S enantiomer of
(3,4)dm8*G1-CTV forms left-handed helical columns.23 spectra of the pure enantiomers of (3,4)dm8*G1-CTV in 4.0 ×
10−5 M dodecane solution upon cooling from 25 to 0 °C are
depicted in Figure 11a. Helical supramolecular polymerization
in this solvent is evidenced by the emergence of a bisignated
CD signal upon cooling. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society The absorbance of 4.0 × 10−5
M solutions of the CTV dendrimers in n-butanol at the
maximum of the main absorption band (206 nm) was recorded
upon cooling from 40 to −10 °C at a slow rate of 0.5 °C/min,
and the data at 206 nm were then converted to the degree of
aggregation Φ (Figure 11c). In all cases (S enantiomer, R
enantiomer, racemic by mixing, and racemic by synthesis CTV)
a sharp transition was observed between the molecularly
dissolved state (Φ = 0) and the start of the self-assembly
process (Φ > 0) with a sudden increase of the degree of
aggregation. This sharp transition indicates a cooperative
nucleation−elongation mechanism. Fitting of the experimental
data with the elongation model from eq 1 generated the values
of Te and he reported in the inset of Figure 11c. p
g
Note that in contrast to the spectra in Figure 11a (which
were recorded in dodecane solution) the measurements
illustrated in Figure 11c were performed in n-butanol solution. Experiments in dodecane gave similar cooling curves, but the
elongation temperatures were low (down to 2 °C for the
racemic by synthesis CTV), which made fitting calculations
inaccurate (especially calculation of he) because the temper-
ature of the CD instrument could only be cooled down to −10
°C and self-assembly was not complete at this temperature. CD
and UV spectra of the self-assembled (3,4)dm8*G1-CTV
derived from enantiomerically pure or racemic samples are
virtually identical in dodecane and n-butanol solutions (Figure
SF6, Supporting Information), which indicates that the same
supramolecular objects are formed in both solvents. Higher
elongation temperatures in n-butanol made calculations more
accurate, and therefore, this solvent was selected for this
experiment. Even though the racemic and achiral dendronized CTV are
CD silent, the UV absorption changes at 206 nm followed upon
temperature changes give insight into the mechanism of self-
assembly of these dendronized CTV as well as their chiral
analogues. The absorption at the band maximum at 206 nm can
be plotted versus temperature to determine whether self-
assembly proceeds via an isodesmic or a cooperative
mechanism following the method developed by the Schenning
and Meijer laboratory.24 In an isodesmic supramolecular
polymerization process the association of each monomer to
another monomer or to a growing chain occurs with the same
equilibrium constant regardless of the length of the chain. Journal of the American Chemical Society By
contrast, a cooperative self-assembly proceeds through two
distinct steps: a nucleation or an activation step characterized
by an equilibrium constant Ka and an elongation or growth step
characterized by a higher equilibrium constant Ke.8i,j,25
Typically nucleation consists of formation of the first turn or
nucleus of the helix. Subsequent growth is facilitated once the
nucleus is formed. The Meijer laboratory determined that
isodesmic and cooperative self-assembly mechanisms can be
distinguished by the curve profile of a temperature-dependent
CD/UV experiment at a fixed wavelength.24 The CD ellipticity
or UV absorbance versus temperature is rescaled between 0 and
1 to reflect the degree of aggregation (Φ) of the self-assembling
dendronized CTV. The curve profile obtained is characteristic
of the mechanism of helical supramolecular polymerization. A
sigmoidal curve corresponds to an isodesmic mechanism,
whereas the presence of a sharp transition at Φ ≈0 is
distinctive of a cooperative mechanism. The transition occurs
when growth of the supramolecular columns starts upon
cooling. The corresponding temperature is referred to as the
elongation temperature Te. The curve of the degree of
aggregation versus temperature can be modeled by eq 1 at
temperatures below Te p
Within experimental error the fitting results for the pure S
enantiomer and for the pure R enantiomer are identical. Helical
supramolecular polymerization of the enantiomerically pure
CTV is an enthalpically driven process with he = −29 ± 1 kcal/
mol that starts at the elongation temperature Te = 29 ± 1 °C in
n-butanol solution at a concentration of 4.0 × 10−5 M. Identical
results are expected for the S and R enantiomers since the only
difference between their self-assembled structures is the helix
handedness. For the pure enantiomers the calculations can also
be made from CD data and give similar results. However, in the
present case the results obtained from UV data were selected
for comparison with the racemic systems, which do not exhibit
any CD signal. When the S and R enantiomers are mixed in equimolar
proportions to form the 50% (S)/50% (R) mixture or racemic
by mixing, the enthalpy for supramolecular polymerization
remains identical to that of pure enantiomers within
experimental error. Journal of the American Chemical Society This thin film CD experiment allows us to compare the helical
columns determined by XRD in the solid state with the self-
assembled structures analyzed by CD/UV spectroscopies in
solution. Self-assembly of the enantiomerically pure CTV in solution is
reflected not only by the emergence of Cotton effects upon
cooling but also by a slight blue shift and significant decrease of
the intensity of the main UV absorption band at 206 nm. These
changes in the UV spectra enable us to monitor the self-
assembly process even in the case of racemic or achiral CTV
dendrimers and to compare it with the self-assembly of
enantiomerically pure (3,4)dm8*G1-CTV. For racemic and
achiral dendronized CTV no Cotton effect is observed upon
cooling, either because the columns are not helical, or because 7179 Journal of the American Chemical Society Article temperature, he is the molar enthalpy for supramolecular
polymerization, and R is the gas constant.8i the columns contain equal proportions of right-handed and left-
handed helical fragments whose CD contributions cancel each
other, or because the columns are helical but exist in an equal
ratio of right- and left-handed columns (Figure SF5, Supporting
Information). Formation of racemic helical columns from a
racemic dendrimer could result from two distinct processes. The first process would be a complete deracemization of the
CTV within columns, with right-handed columns composed
exclusively of R enantiomers and left-handed columns
composed exclusively of S enantiomers. The second process
would be a partial deracemization of the CTV to form
enantiomerically rich but not enantiomerically pure columns. These columns would contain an excess of one enantiomer,
which would dictate the helical sense of the columns. As
discussed previously, DSC and XRD experiments demonstrated
that in the solid state the 50% (S)/50% (R) mixture forms
helical columns whose enantiomeric purity can be enhanced by
annealing at 60 °C. These results suggest that the columns
formed in solution are helical, with equal contributions from
left- and right-handed columns canceling each other. The
enantiomeric purity of the columns in solution was further
assessed in a majority-rules experiment to be discussed in a later
subsection. g
The method described above was used to determine the
mechanism of self-assembly of chiral and racemic (3,4)-
dm8*G1-CTV and the thermodynamics of their helical
supramolecular polymerization. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society Consequently,
data from cooling scans were exclusively used for fitting with
the elongation equation and calculation of the enthalpy for
supramolecular polymerization. columns formed from pure enantiomers since they have
matching DSC traces and XRD results. The constant enthalpy
he observed for the 50% (S)/50% (R) mixture compared to
pure enantiomers as well as the weakness of the majority-rules
effect (discussed later) suggest that this deracemization process
occurs in solution as well as in the solid state, although it is not
perfect in solution. In this context deracemization occurs upon
self-assembly, which means that the elongation is mostly
homochiral (S enantiomers self-assemble with S enantiomers to
form left-handed helical columns, and R enantiomers self-
assemble with R enantiomers to form right-handed columns). Even if self-sorting is not perfect in solution, the enthalpy
results and majority-rules experiment suggest that it is
reasonable to assume that homochiral elongation is the
predominant elongation mode. The self-sorting process implies
that the effective concentration of each enantiomer available to
build each type of column (left-handed or right-handed) in
solution is only one-half of the total concentration of
dendrimers. Therefore, in order to directly compare the
elongation temperature of the 50% (S)/50% (R) mixture
with the pure enantiomers, we need to consider a solution of
the racemic mixture with a total concentration of 8.0 × 10−5 M,
which corresponds to an effective concentration of 4.0 × 10−5
M for each enantiomer (identical to the concentration of the
solutions of pure enantiomers used to calculate the elongation
parameters). At this concentration we calculate an elongation
temperature of 27 °C (Figure 11c) vs 28 °C for the pure R
enantiomer and 29 °C for the pure S enantiomer. This result is
consistent with the hypothesis that the enantiomers deracemize
in solution, but self-sorting is not perfect and leads to a slight
decrease of Te. p
p y
It is also important to note that eq 1 is based on a helical self-
assembly model that involves only supramolecular polymer-
ization of identical monomers. This model is suitable for the
enantiomerically pure dendronized CTV. However, self-
assembly of the racemic systems (racemic by mixing and
racemic by synthesis) does not exactly correspond to the type
of system for which the model was developed. Journal of the American Chemical Society However, the elongation temperature of
the racemic by mixing at a total dendrimer concentration of 4.0
× 10−5 M is lower than that of the enantiomerically pure self-
assemblies: fitting calculations give Te = 23 °C for the racemic
by mixing vs 28−29 °C for the pure enantiomers at the same
concentration (Figure SF4, Supporting Information). These
fitting results may be rationalized in light of the characterization
of the 50% (S)/50% (R) mixture in the solid state. Annealing
experiments revealed that the thermodynamic product in bulk
consists of enantiomerically pure columns that are identical to Φ = Φ
−
−
−
⎡
⎣
⎢
⎢
⎛
⎝
⎜
⎞
⎠
⎟
⎤
⎦
⎥
⎥
h
RT
T
T
1
exp
(
SAT
e
e
2
e
(1) (1) where Φ is the degree of aggregation, ΦSAT is the saturation
level of the data, T is the temperature, Te is the elongation 7180 Journal of the American Chemical Society Article Article heating, whereas the cooling curves reflect the thermodynamic
self-assembly process. This hypothesis was confirmed by two
“temperature-hold” experiments. In a first experiment, a 8.0 ×
10−5 M solution of (S)-(3,4)dm8*G1-CTV was cooled down
from 50 to 25 °C (which is the center temperature of the
hysteresis cycle), whereupon the temperature was held, and CD
changes with time were monitored at 240 nm. The ellipticity
did not change over time and remained close to zero ellipticity
(0 mdeg), which confirms that the thermodynamic state of the
system at this temperature is the molecularly dissolved state
and not the self-assembled state. In a second experiment, the
solution was heated at a rate of 0.5 °C/min from the self-
assembled state at 0−25 °C, whereupon the temperature was
held. Slow changes in the CD signal were observed over time,
and the ellipticity slowly decreased to reach nearly 0 mdeg after
90 h (Figure SF7, Supporting Information). The decrease in
ellipticity reflects a slow dissociation of the self-assembled
columns over time, which demonstrates that the self-assembled
state is thermodynamically unstable at 25 °C but the kinetics of
dissociation are very slow. Overall, the temperature-hold
experiments indicate that cooling scans reflect the thermody-
namic behavior of the system, while the data obtained from
heating scans are affected by kinetic effects and are therefore
not suitable for thermodynamic calculations. Journal of the American Chemical Society For example,
supramolecular polymerization of the racemic by mixing
involves two distinct monomers (the S and R enantiomers),
which can potentially form different types of assemblies, right-
handed and left-handed helical columns or columns with no
well-defined helicity. However, the two types of monomers
differ only in the stereochemistry of their peripheral alkyl
chains, and their self-assembly involves many supramolecular
interactions over the whole surface of the dendronized CTV. Therefore, we can reasonably assume that the differences
between the equilibrium constants of monomer additions to
growing chains are small compared to the order of magnitude
of the equilibrium constants. In other words, we assume that
the equilibrium constants are approximately the same for each
monomer addition to each type of column, and we use the
same elongation model in all cases. This assumption is
supported by the good fit between the experimental curve
profile and the model. Calculations for the self-assembly of the mixture of
stereoisomers racemic by synthesis-(3,4)dm8*G1-CTV give
he = −13 kcal/mol and Te = 18 °C. For this system both the
enthalpy and the elongation temperature are lower than those
of the pure enantiomers and 50% (S)/50% (R) mixture. These
characteristics in solution are in good agreement with the
conclusions of the solid state experiments. XRD and DSC of
the racemic by synthesis sample showed significant loss of order
in the 3D periodic array of supramolecular columns, which melt
at a much lower temperature. These observations are in
accordance with the lower enthalpy and elongation temperature
obtained for the racemic by synthesis in solution. The loss of
order in the self-assembled structure is demonstrated by the
fact that columns of the racemic by synthesis dendronized CTV
are less thermally stable than the columns formed by the pure
enantiomers or the racemic by mixture and that their formation
is not as enthalpically favorable. Moreover, the clear differences
observed between he and Te of the racemic by synthesis CTV
and the 50% (S)/50% (R) mixture confirm that the
enantiomerically pure compounds play an important role in
facilitating and stabilizing formation of helical supramolecular
columns. In order to gain more insight into the thermodynamics of the
helical supramolecular polymerization, temperature-dependent
CD/UV experiments were performed at different concen-
trations in n-butanol. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society However, the clear trend observed for the elongation
temperature, which decreases from the pure enantiomers to
the racemic by mixing to the racemic by synthesis,
demonstrates the differences in thermal stability between
these systems and proves that chirality facilitates the self-
assembly of dendronized CTV. van’t Hoffplot is higher than the value obtained by fitting of the
cooling data at 4.0 × 10−5 M with the elongation equation (he =
−29 kcal.mol−1). This difference and dependence on the
wavelength used for calculation discussed in the Supporting
Information indicate that the enthalpy and entropy cannot be
determined with great precision using these methods. However,
the sign and order of magnitude of the enthalpy obtained using
both methods are the same and correspond to the expected
thermodynamic behavior. The respective signs of the enthalpy
and entropy indicate that the self-assembly of dendronized
CTV molecules is an enthalpically driven process. Supra-
molecular polymerization is enthalpically favored and entropi-
cally disfavored. The enthalpic contribution predominates
below the elongation temperature Te, while the entropic
contribution promotes dissociation of the columns above Te. van’t Hoffplots were also obtained for both racemic systems
(the racemic by mixing and the racemic by synthesis
(3,4)dm8*G1-CTV). The results were similar to those
obtained for the S enantiomer, which suggests that the
differences in enthalpy and entropy between these systems
are too small to be accurately determined from van’t Hoffplots. However, the clear trend observed for the elongation
temperature, which decreases from the pure enantiomers to
the racemic by mixing to the racemic by synthesis,
demonstrates the differences in thermal stability between
these systems and proves that chirality facilitates the self-
assembly of dendronized CTV. p
p
Microspot CD Analysis of Thin Films. Although almost
no net helicity was observed in racemized samples obtained by
mixing or by synthesis in solution by CD measurements, it is of
interest to understand whether the self-assemblies are race-
mized within the column or in between columns as already
suggested by XRD results, that is, we wish to determine
whether individual supramolecular columns are helical and
single handed but Cotton effects are macroscopically canceled
due to opposite contributions or individual columns are
racemized along the column and have poorly defined helicities. Since solution CD experiments only provide averaged
information within a relatively large solution volume, signals
from individual columns are not accessible. Journal of the American Chemical Society In order to obtain
chiral information from monodomains that might show
preference of helical sense, a microspot CD setup equipped
with ultrasmall beam size, less than a few hundred micrometers,
was used to examine solid state thin films made of racemic and
achiral samples (Figure 12). Thin films of fully racemized or
racemic by synthesis (3,4)dm8*G1-CTV and achiral (3,4)8G1-
CTV with linear 8-carbon side chains were prepared on a
quartz plate by spin coating from 2% w/v chloroform solutions
followed by melting at 85 °C and slow cooling to RT for
development of the Φh
k structure. Note that the birefringence
of these films observed by polarized microscopy was confirmed Majority-Rules Experiment. A majority-rules experi-
ment26 was performed (Figure 11d and 11e) in order to
elucidate the effect of mismatched chirality on the self-assembly
of CTV dendrimers. This experiment consists of comparing the
Cotton effects of solutions that contain various proportions of S
and R units in a helical polymer or supramolecular polymer. In
the absence of a majority-rules effect the ratio of left-handed vs
right-handed helices should be identical to the ratio of S vs R
enantiomers and a linear response is expected. The ellipticity at
a given wavelength is proportional to the enantiomer excess. However, if the system exhibits a majority-rules effect, a
nonlinear response is observed and the ellipticity should exceed
the expected linear behavior. This nonlinear effect occurs if the
major enantiomer can dictate its preferred helical sense to the
minor enantiomer and the “wrong” enantiomer can be inserted
into a helical supramolecular column without disturbing its
helicity. In order to investigate the majority-rules effect in the
self-assembly of CTV dendrimers, CD spectra of solutions of
(S)- and (R)-(3,4)dm8*G1-CTV (total concentration of 4.0 ×
10−5 M in dodecane) mixed in various proportions were
measured at 0 °C after slow cooling (0.5 °C/min) from the
molecularly dissolved state at 50 °C. The CD spectra and net
helicity (obtained from the ellipticity at 289 nm) vs
enantiomeric excess are depicted in Figure 11d and 11e. Although the effect is not very intense under these conditions
in this solvent, we observe a deviation from linearity and the net
helicity slightly outpaces the enantiomeric excess. As discussed
in previous sections of this study, mixtures of enantiomers of
dendronized CTV tend to deracemize to form enantiomerically
pure columns. Journal of the American Chemical Society Determining the elongation temperature
Te at different concentrations of dendronized CTV allowed
calculation of both the enthalpy he and the molar entropy se
that characterize the elongation step in the cooperative self-
assembly process. These thermodynamic parameters were
obtained from a van’t Hoffplot.8i For example, the data
obtained for the pure S enantiomer of (3,4)dm8*G1-CTV gave
he = −44 kcal·mol−1 and se = −126 cal·mol−1·K−1 (Figure SF8,
Supporting Information). The value of he calculated from the The temperature-dependent CD/UV experiments were
performed at a slow cooling rate of 0.5 °C/min to ensure
that the system always remained at thermodynamic equilibrium. However, a hysteresis was systematically observed between
cooling and heating cycles. Attempts to slow down the heating/
cooling rate did not eliminate the hysteresis, even at 0.08 °C/
min. However, it was observed that the heating curves are
affected by the heating rate, while the cooling curves remained
essentially unaffected. This result suggests that kinetic effects
affect the dissociation of the self-assembled columns upon 7181 Journal of the American Chemical Society Article discussed results on deracemization but indicates that this
process is not complete in solution. van’t Hoffplot is higher than the value obtained by fitting of the
cooling data at 4.0 × 10−5 M with the elongation equation (he =
−29 kcal.mol−1). This difference and dependence on the
wavelength used for calculation discussed in the Supporting
Information indicate that the enthalpy and entropy cannot be
determined with great precision using these methods. However,
the sign and order of magnitude of the enthalpy obtained using
both methods are the same and correspond to the expected
thermodynamic behavior. The respective signs of the enthalpy
and entropy indicate that the self-assembly of dendronized
CTV molecules is an enthalpically driven process. Supra-
molecular polymerization is enthalpically favored and entropi-
cally disfavored. The enthalpic contribution predominates
below the elongation temperature Te, while the entropic
contribution promotes dissociation of the columns above Te. van’t Hoffplots were also obtained for both racemic systems
(the racemic by mixing and the racemic by synthesis
(3,4)dm8*G1-CTV). The results were similar to those
obtained for the S enantiomer, which suggests that the
differences in enthalpy and entropy between these systems
are too small to be accurately determined from van’t Hoffplots. ■CONCLUSIONS However, when deracemiza-
tion is accessible, it can transform mixtures of left-handed and
right-handed columns containing different enantiomeric
purities including racemic and achiral into highly ordered
single domains of enantiopure single-handed columns. Homochiral supramolecular polymerization of racemic mixtures
was previously reported only in solution for rigid S-shape and
rigid macrocycles as well as for disc-like compounds with an
exceedingly high number of stereogenic centers.8d−f However,
spontaneous deracemization or chiral self-sorting of supra-
molecular columns obtained from various enantiomeric ratios
including racemic and from achiral building blocks, to our best
knowledge, has been demonstrated for the first time in their
crystal state in this publication. Elucidating the molecular
principles to realize chiral self-sorting at the supramolecular
level in the crystal state will have numerous technological Similar microspot CD experiments were also conducted on a
thin film of achiral (3,4)8G1-CTV with linear achiral 8-carbon
side chains (Figure 12b). Surprisingly, although the intensity
was even weaker than its chiral analogue, positive or negative
net ellipticity was still observed from spot to spot. This suggests
that the spontaneous deracemizing of supramolecular columns
occurs even in achiral CTVs, and the overall film is racemic
because of the mixing of right- and left-handed columns. The
even weaker CD intensities indicate the random distribution of
right- and left-handed columns throughout the film, and the
monodomain size is much smaller than the beam size. The
random distribution of columns results in less perfect crystals at
room temperature as evidenced by the observation of fewer and
weaker quadrant reflections in XRD patterns. q
p
From the above discussion, we concluded that all CTV
samples self-assemble into chiral nonracemic supramolecular
columns since even achiral compound forms domains with
single chirality. Microphase separation of columns with
different handedness generates separated crystalline domains. Therefore, racemic samples are racemized between domains,
not within each column. However, the phase separation
strength is not strong in the bulk state and results in small
domain sizes that give weak CD intensities. The handedness is
determined at an early stage of column formation, the
nucleation step. Once the handedness is decided by the first
few molecules, the next molecule will follow the same rotation
sense during the elongation step even if the rotation sense is
not preferred by the molecule, since making an opposite
rotation in an existing column would cost more energy and is
not favored. ■CONCLUSIONS Since Φh
k monodomains can be
generated only from columns of single handedness, the order of
the Φh
k crystals is determined by the enantiomeric purity of
their single-handed columns. Therefore, deracemization of
single-handed columns containing different enantiomeric
compositions, including racemic, could be monitored by
annealing in the crystal state at 60 °C. Solid state NMR
experiments demonstrated that column deracemization occurs
via the exit and reentry of the disc-like transient conformer of
the hat-shaped dendronized CTV that is available during the
crown to crown inversion. Overall, these results reveal that
supramolecular chirality, at least in columnar architectures, can
be generated from racemic and even achiral building blocks. They also demonstrate again1c,12 that when chirality is present
homochirality is essential in formation of highly ordered, stable,
and 3D Φh
k crystalline assemblies. However, when deracemiza-
tion is accessible, it can transform mixtures of left-handed and
right-handed columns containing different enantiomeric
purities including racemic and achiral into highly ordered
single domains of enantiopure single-handed columns. Homochiral supramolecular polymerization of racemic mixtures
was previously reported only in solution for rigid S-shape and
rigid macrocycles as well as for disc-like compounds with an
exceedingly high number of stereogenic centers.8d−f However,
spontaneous deracemization or chiral self-sorting of supra-
molecular columns obtained from various enantiomeric ratios
including racemic and from achiral building blocks, to our best
knowledge, has been demonstrated for the first time in their
t l
t t
i
thi
bli ti
El
id ti
th
l
l Synthesis and self-organization of a library based on five self-
assembling dendronized cyclotriveratrylenes (CTV) with R and
S side chain chirality containing various combinations of R with
S including racemic by mixing and racemic by synthesis as well
as achiral analogues with n-octyl and n-dodecyl side chains was
reported. The structures of all assemblies generated from these
dendronized CTV that are shape persistent in solution and in
bulk were determined by a combination of complementary
techniques including CD-UV in solution and thin film,
microspot CD in thin film, DSC, fiber XRD, and computer
simulation of molecular models of the oriented fiber XRD to
reveal the mechanisms of self-assembly, racemization, and
deracemization during their helical supramolecular polymer-
ization in solvophobic solvents and in their 3D Φh
k and 2D Φh
phases. ■CONCLUSIONS ■CONCLUSIONS to be negligibly small. Each curve in Figure 12 represents a
single measurement at one microspot on the film. The signal
from wavelengths below 200 nm is affected by the quartz
substrate and shows irregular patterns that should be ignored. Signals from the dendronized CTVs are located between 200
and 250 nm. Figure 12a shows the results from the film made of
racemic by synthesis sample. Here the 12 side chains with
either R or S chiral center are randomly arranged on each
molecule and lead to no preference of rotation sense during
column assembly and therefore show zero net helicity in
solution CD measurements. Synthesis and self-organization of a library based on five self-
assembling dendronized cyclotriveratrylenes (CTV) with R and
S side chain chirality containing various combinations of R with
S including racemic by mixing and racemic by synthesis as well
as achiral analogues with n-octyl and n-dodecyl side chains was
reported. The structures of all assemblies generated from these
dendronized CTV that are shape persistent in solution and in
bulk were determined by a combination of complementary
techniques including CD-UV in solution and thin film,
microspot CD in thin film, DSC, fiber XRD, and computer
simulation of molecular models of the oriented fiber XRD to
reveal the mechanisms of self-assembly, racemization, and
deracemization during their helical supramolecular polymer-
ization in solvophobic solvents and in their 3D Φh
k and 2D Φh
phases. Supramolecular columns self-assemble in solution
through a cooperative nucleation−elongation mechanism with
enthalpies ranging from 29 kcal/mol for enantiopure and
racemic by mixture dendronized CTV to 18 kcal/mol for
racemic by synthesis CTV. These values are lower than those of
previously investigated dendritic dipeptides that range from 35
to 19 kcal/mol and did not undergo deracemization in bulk.1c,12
The enantiopure dendronized CTVs self-assemble into single-
handed columns, while racemic mixtures are generated from
equal ratios of left- and right-handed supramolecular columns
that can be considered a homochiral supramolecular polymer-
ization.8d,e No intracolumnar racemization was detected in any
of these chiral, chiral racemic, or achiral compositions; in other
words, the self-assembled columns are single-handed helical in
all cases. Even columns generated from achiral dendronized
CTV and racemic by synthesis CTV exhibit single handedness
in their Φh
k monodomains. ■CONCLUSIONS Supramolecular columns self-assemble in solution
through a cooperative nucleation−elongation mechanism with
enthalpies ranging from 29 kcal/mol for enantiopure and
racemic by mixture dendronized CTV to 18 kcal/mol for
racemic by synthesis CTV. These values are lower than those of
previously investigated dendritic dipeptides that range from 35
to 19 kcal/mol and did not undergo deracemization in bulk.1c,12 Nevertheless, opposite signs of ellipticity were observed at
different microspots in the thin film (Figure 12a). This
confirms formation of monodomains consisting of right- and
left-handed columns and suggests that a spontaneous
deracemization process involves diffusion between columns
and that the column handedness is most probably decided at
the nucleation step. Although the columns seem to possess
selected handedness, the phase separation of columns with
opposite handedness is weak and therefore results in very small
domain size. The mixing of right- and left-handed columns
prevents formation of 3D ordered crystals and generates both
inter- and intracolumn packing defects as evidenced by very
weak CD signals and low melting temperatures found in the
DSC of the as-prepared samples. 9
/
g
The enantiopure dendronized CTVs self-assemble into single-
handed columns, while racemic mixtures are generated from
equal ratios of left- and right-handed supramolecular columns
that can be considered a homochiral supramolecular polymer-
ization.8d,e No intracolumnar racemization was detected in any
of these chiral, chiral racemic, or achiral compositions; in other
words, the self-assembled columns are single-handed helical in
all cases. Even columns generated from achiral dendronized
CTV and racemic by synthesis CTV exhibit single handedness
in their Φh
k monodomains. Since Φh
k monodomains can be
generated only from columns of single handedness, the order of
the Φh
k crystals is determined by the enantiomeric purity of
their single-handed columns. Therefore, deracemization of
single-handed columns containing different enantiomeric
compositions, including racemic, could be monitored by
annealing in the crystal state at 60 °C. Solid state NMR
experiments demonstrated that column deracemization occurs
via the exit and reentry of the disc-like transient conformer of
the hat-shaped dendronized CTV that is available during the
crown to crown inversion. Overall, these results reveal that
supramolecular chirality, at least in columnar architectures, can
be generated from racemic and even achiral building blocks. They also demonstrate again1c,12 that when chirality is present
homochirality is essential in formation of highly ordered, stable,
and 3D Φh
k crystalline assemblies. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 Journal of the American Chemical Society If the deracemization process was perfect then
no majority-rules effect should be observed because left-handed
columns should contain exclusively S enantiomers and right-
handed columns exclusively R enantiomers. Therefore, the ratio
of left- vs right-handed columns, and hence the net helicity,
should be proportional to the enantiomeric excess. The weak
majority-rules effect observed here supports previously Figure 12. Thin film micro-CD spectra of (a) racemic by synthesis
(3,4)dm8*G1-CTV and (b) (3,4)8G1-CTV with achiral 8-carbon
linear side chains collected in the Φh
k phase at room temperature. Films were spin coated on a quartz plate followed by melting at 85 °C
and subsequent cooling to room temperature. Each spectrum was
obtained from different spots on the film. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185
2 Figure 12. Thin film micro-CD spectra of (a) racemic by synthesis
(3,4)dm8*G1-CTV and (b) (3,4)8G1-CTV with achiral 8-carbon
linear side chains collected in the Φh
k phase at room temperature. Films were spin coated on a quartz plate followed by melting at 85 °C
and subsequent cooling to room temperature. Each spectrum was
obtained from different spots on the film. dx.doi.org/10.1021/ja5035107 | J. Am. Chem. Soc. 2014, 136, 7169−7185 7182 Journal of the American Chemical Society Article Article ■CONCLUSIONS This gives again insight
into the reasons why nature selected and retained homochir-
ality during its evolution. Notes The authors declare no competing financial interest. The authors declare no competing financial interest. 10806. (h) van Gestel, J.; Palmans, A. R. A.; Titulaer, B.; Vekemans, J. A. J. M.; Meijer, E. W. J. Am. Chem. Soc. 2005, 127, 5490−5494. ■ACKNOWLEDGMENTS (i) Jonkheijm, P.; van der Schoot, P.; Schenning, A. P. H. J.; Meijer, E. W. Science 2006, 313, 80−83. (j) Percec, V.; Ungar, G.; Peterca, M. ( ) Financial support by the National Science Foundation (DMR-
1066116 and DMR-1120901), the Humboldt Foundation, and
the P. Roy Vagelos Chair at Penn (all to V.P.) is gratefully
acknowledged. G.U. and X.Z. acknowledge support from the
joint NSF-EPSRC PIRE project “RENEW” (EPSRC grant EP-
K034308). Science 2006, 313, 55−56. (k) Korevaar, P. A.; George, S. J.;
Markvoort, A. J.; Smulders, M. M. J.; Hilbers, P. A. J.; Schenning, A. P. g
H. J.; De Greef, T. F. A.; Meijer, E. W. Nature 2012, 481, 492−496. ( ) (l) Pèrez-Garcìa, L.; Amabilino, D. B. Chem. Soc. Rev. 2007, 36, 941−
967. (m) Fasel, R.; Parschau, M.; Ernst, K.-H. Nature 2006, 439, 449− 452. (n) Safont-Sempere, M. M.; Fernández, G.; Würthner, F. Chem. Rev. 2011, 111, 5784−5814. (o) Fletcher, S. P.; Jagt, R. B. C.; Feringa, ■CONCLUSIONS Therefore, the supramolecular columns are not
enantiomerically pure but are of single handedness. The
molecules forced to stack in columns with nonfavored rotation
will not be able to pack perfectly with molecules in neighboring
columns, creating defects in the crystal by destroying the
correlation between columns. The melting temperature is
determined by the defect density in the crystal. 7183 Journal of the American Chemical Society Article G.; Batty, S. V. J. Chem. Soc., Perkin Trans. 1 1993, 1411−1420. (g) Percec, V.; Heck, J.; Tomazos, D.; Falkenberg, F.; Blackwell, H.;
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structure mediates functions. Finally, a parallel can be drawn
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building blocks lead to more stable, more ordered, and thus
more perfectly functional structures. ■ASSOCIATED CONTENT Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10801−
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УДК 339.924 : 338.45 Кушніренко О. М.,
Зарудна О. С. Зарудна О. С. Актуальність теми дослідження пояснюється впливом глобалізації в результаті якої все більше
і більше продуктів проходять через глобальні ланцюги доданої вартості, щоб досягти кінцевого
споживача, що призводить до появи нових форм організації суспільного виробництва, включення до яких
надає нові можливості для українських виробників. Постановка проблеми. Використання сприятливих можливостей та мінімізація загрозливих
чинників лібералізації зовнішньої торгівлі у все більш відкритому та конкурентному середовищі
ставить нові вимоги для виробничих підприємств. Розвиток глобальних виробничих систем надає
можливості для включення в глобальні ланцюжки доданої вартості промисловості України та потребує
подальших наукових досліджень. Аналіз останніх досліджень і публікацій. Різні аспекти розвитку глобальних ланцюгів доданої
вартості промисловості знайшли відображення у багатьох наукових працях зарубіжних та вітчизняних
учених: П.Марша, Р. Каплінського, К.Шваба, Р.Райха, Д. Родріка, Е.Райнерта, С.Вебера, Г.Джереффі,
Гейця В.М., Вишневського О.С., Дейнеко Л.В., Кизима М.О., Кваша Т.К., Ляшенко В.І., Мусіної Л.А.,
Пятницького В.Т., Сіденко В.Р. та інших. Виділення недосліджених частин загальної проблеми. В умовах посилення впливу інтеграційних
процесів на розвиток міжнародної торгівлі та виробництва залишається недостатньо дослідженою
проблема вибору найбільш ефективних способів інтеграції до глобальних ланцюгів доданої вартості для
переробної промисловості України як надійного постачальника продукції з більш високим ступенем переробки. Постановка завдання, мети дослідження. Завданням дослідження є аналіз особливостей
формування ланцюгів створення доданої вартості та способи інтеграції до них для переробної
промисловості України як надійного постачальника продукції з більш високим ступенем переробки. Метою дослідження є розробка пропозицій щодо можливих напрямів сприяння інтеграції переробної
промисловості України до глобальних ланцюгів створення доданої вартості. Метод або методологія проведення дослідження. У роботі використані загальнонаукові методи:
абстрактно-логічний, індукції та дедукції, системного підходу; аналізу і синтезу, а також спеціальні
методи дослідження: структурно-функціонального аналізу, експертних оцінок. Метод або методологія проведення дослідження. У роботі використані загальнонаукові методи:
абстрактно-логічний, індукції та дедукції, системного підходу; аналізу і синтезу, а також спеціальні
методи дослідження: структурно-функціонального аналізу, експертних оцінок. Викладення основного матеріалу (результати роботи). Розкрито існуючі підходи до визначення
поняття ланцюгів створення доданої вартості та здійснено їх групування за сутнісними ознаками;
досліджено особливості та сучасні тренди розвитку глобальних ланцюгів вартості в переробній
промисловості; виокремлено можливості та існуючі ризики при вбудовуванні в ланцюги створення
доданої вартості для країн, що розвиваються; та обґрунтовано практичні рекомендації щодо вибору
найбільш ефективних способів інтеграції до глобальних ланцюгів доданої вартості для переробної
промисловості України як надійного постачальника продукції з більш високим ступенем переробки. р
р
р
у
у
р р
Галузь застосування результатів. УДК 339.924 : 338.45 Результати цього дослідження можуть бути застосовані в
процесі формування державної промислової політики щодо основних аспектів розвитку промисловості
в контексті інтеграційних процесів. Висновки відповідно до статті За результатами проведеного дослідження було здійснено оцінку
інтеграції українських виробничих компаній в міжнародні ланцюги доданої вартості. Враховуючи зміну
географічних та товарних векторів вітчизняного експорту, подальші перспективи участі України в
глобальному торгівельному просторі залежатимуть від того, чи буде досягнуто зростання
конкурентоспроможності внутрішньої частини ланцюга доданої вартості. Впровадження ефективних
інструментів стимулювання входження переробної промисловості України до глобальних ланцюгів
доданої вартості передбачає створення ефективної політики та інституцій, націлених на усунення
обмежень при інтеграції українського виробника до міжнародних виробничих мереж. ва: міжнародна торгівля, торгівельна політика, промисловість, глобальні ланцюги
ті, експортний потенціал, торгівельна інтеграція. чові слова: міжнародна торгівля, торгівельна політика, промисловість, глобальні л
вартості, експортний потенціал, торгівельна інтеграція. ВОЗМОЖНОСТИ ИНТЕГРАЦИИ УКРАИНСКОЙ ПРОМЫШЛЕННОСТИ ВОЗМОЖНОСТИ ИНТЕГРАЦИИ УКРАИНСКОЙ ПРОМЫШЛЕННОСТИ
Й В ГЛОБАЛЬНЫЕ ЦЕПОЧКИ ДОБАВЛЕННОЙ СТОИМОСТИ ЕКОНОМІКА ТА УПРАВЛІННЯ
УДК 339.924 : 338.45
https://doi.org/10.31470/2306-546X-2018-39-65-74
МОЖЛИВОСТІ ІНТЕГРАЦІЇ УКРАЇНСЬКОЇ ПРОМИСЛОВОСТІ
ДО ГЛОБАЛЬНИХ ЛАНЦЮГІВ ДОДАНОЇ ВАРТОСТІ ЕКОНОМІКА ТА УПРАВЛІННЯ Кушниренко О. Н.,
Зарудная О. С. руд
Актуальность темы исследования объясняется влиянием глобализации в результате которой
все больше и больше продуктов проходят через глобальные цепи добавленной стоимости при
поступлении к конечному потребителю, что приводит к появлению новых форм организации
общественного производства, включение в которые предоставляет новые возможности для
украинских производителей. © Кушніренко О. М., Зарудна О. С., 2018 Економічний вісник університету | Випуск № 39 65 ЕКОНОМІКА ТА УПРАВЛІННЯ Постановка проблемы. Использование благоприятных возможностей и минимизация угрожающих
факторов либерализации внешней торговли во все более открытой и конкурентной среде ставит
новые требования для производственных предприятий. Развитие глобальных производственных
систем предоставляет возможности для включения в глобальные цепочки добавленной стоимости
для промышленности Украины и требует дальнейших научных исследований. Анализ последних исследований и публикаций. Различные аспекты развития глобальных цепочек
добавленной стоимости промышленности нашли отражение во многих научных трудах зарубежных и
отечественных ученых: П.Марша, Р. Каплински, К.Шваба, Р.Райха, Д. Родрика, Е.Райнерта, С.Вебера,
Г.Джереффи, Гейца В.М., Вишневского А.С., Дейнеко Л.В., Кизима Н.А., Кваши Т.К., Ляшенко В.И.,
Мусиной Л.А., Пятницкого В.Т., Сиденко В.Р. и других. Анализ последних исследований и публикаций. Различные аспекты развития глобальных цепочек
добавленной стоимости промышленности нашли отражение во многих научных трудах зарубежных и
отечественных ученых: П.Марша, Р. Каплински, К.Шваба, Р.Райха, Д. Родрика, Е.Райнерта, С.Вебера,
Г.Джереффи, Гейца В.М., Вишневского А.С., Дейнеко Л.В., Кизима Н.А., Кваши Т.К., Ляшенко В.И.,
Мусиной Л.А., Пятницкого В.Т., Сиденко В.Р. и других. Выделение неисследованных частей общей проблемы. В условиях усиления влияния
интеграционных процессов на развитие международной торговли и производства проблема выбора
наиболее эффективных способов интеграции в глобальные цепочки добавленной стоимости для
перерабатывающей промышленности Украины как надежного поставщика продукции с более высокой
степенью переработки остается недостаточно исследованной. Постановка задачи, цели исследования. Задачей исследования является анализ особенностей
формирования цепей создания добавленной стоимости и способы интеграции в них для
перерабатывающей промышленности Украины как надежного поставщика продукции с более высокой
степенью переработки. Целью исследования является разработка предложений относительно
возможных направлений содействия интеграции перерабатывающей промышленности Украины в
глобальные цепочки добавленной стоимости. Метод или методология проведения исследования. В работе использованы общенаучные
методы: абстрактно-логический, индукции и дедукции, системного подхода; анализа и синтеза, а
также специальные методы исследования: структурно-функционального анализа, экспертных оценок. Метод или методология проведения исследования. В работе использованы общенаучные
методы: абстрактно-логический, индукции и дедукции, системного подхода; анализа и синтеза, а
также специальные методы исследования: структурно-функционального анализа, экспертных оценок. Изложение основного материала (результаты работы). Кушниренко О. Н.,
Зарудная О. С. Раскрыты существующие подходы к
определению понятия цепочек добавленной стоимости и осуществлена их группировка по
существенным признакам; исследованы особенности и современные тренды их развития в
перерабатывающей промышленности; выделены возможности и существующие риски при
встраивании в цепочки добавленной стоимости для развивающихся стран и обоснованы практические
рекомендации по выбору наиболее эффективных способов интеграции в глобальные цепочки
добавленной стоимости для перерабатывающей промышленности Украины. Область применения результатов Результаты этого исследования могут быть применены в
процессе формирования государственной промышленной политики по основным аспектам развития
промышленности в контексте интеграционных процессов. Выводы в соответствии со статьей. По результатам проведенного исследования была
проведена оценка интеграции украинских производственных компаний в международные цепи
добавленной стоимости. Учитывая изменение географических и товарных векторов отечественного
экспорта, дальнейшие перспективы участия Украины в глобальном торговом пространстве зависеть
от того, будет ли достигнут рост конкурентоспособности внутренней части цепочки добавленной
стоимости. Внедрение эффективных инструментов стимулирования вхождения перерабатывающей
промышленности Украины в глобальные цепочки добавленной стоимости предусматривает создание
эффективной политики и институтов, нацеленных на устранение ограничений при интеграции
украинского производителя с международными производственных сетей. Ключевые слова: международная торговля, торговая политика, промышленнос
цепочки добавленной стоимости, экспортный потенциал, торговая интеграция. Економічний вісник університету | Випуск № 39 OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS Kushnirenko О. М., Kushnirenko О. М.,
Zarudna О. S. ,
Zarudna О. S. Relevance of the research topic is due to the impact of globalization which had brought an increasing number
of more and more products pass through global value-added chains to reach the end users. That has led to new
forms of transnational production, that gives new opportunities for Ukrainian producers. p
g
pp
p
Formulation of the problem. The utilizing the opportunities and mitigating the negative impact of the
liberalization of foreign trade makes new requirements for production oriented businesses in a more open and
competitive international environment. The development of global production systems provides opportunities for
participating in global value chains, that opens up new opportunities for the industry of Ukraine and requires further
scientific researches. Analysis of recent research and publications. There are various dimensions to the development of global
value chains of industry that need to be taken into account. The most important of these are P.Marsh, R.Kaplinski
and Morris, K.Schwab, R.Rajk, D.Rodrik, E.Rajnert, S.Veber, P Labasta, G. Dzerffi, Geets VM, Vishnevsky AS,
Deineko LV, Kizim NA, Kvasha TK, Lyashenko VI, Musina LA, Pyatnitsky VT, Sidenko V.R. and others. Економічний вісник університету | Випуск № 39 66 ЕКОНОМІКА ТА УПРАВЛІННЯ Selection of unexplored parts of the general problem. In the context of increasing influence of integration
processes on the development of international trade and production, the problem of choosing the most effective
ways of integration into global value chains for the processing industry of Ukraine as a reliable supplier of products
with a higher degree of processing remains insufficiently studied. Setting the task, the purpose of the study. The objective of the article is to analyze the features of the formation
of value added chains and ways of integrating them into the processing industry of Ukraine as a reliable supplier of
products with a higher degree of processing. The purpose of the study is developing the proposals for possible
constructive ways of promoting the integration of Ukrainian processing industry into global value-added chains. y
p
g
g
p
g
y
g
Method or methodology for conducting research. This paper used of general scientific: abstract-logical,
induction and deduction, systemic approach; analysis, and synthesis and special scientific research methods:
statistical comparisons, grouping, sampling; structure-functional analysis, expert judgments. OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS Presentation of the main material (results of work).The existing approaches to the definition of the notion
of value added chains are disclosed and their grouping is carried out on significant grounds; The features and
modern trends in the development of global value chains in the processing industry are explored; The
opportunities and the existing risks have been identified in the chain of value added for the developing countries;
and substantiated practical recommendations for choosing the most effective ways of integration into global value
chains for the processing industry of Ukraine as a reliable supplier of products with a higher degree of processing. The field of application of results.. The results of this research can be applied in the process of formation
and implementation of Ukraine's integration industrial policy. Conclusions according to the article. In the article authors was made in assessment of the integration of
Ukrainian manufacturing companies into global value chains. The adoption of effective tools and instruments for
encouraging the entry of Ukraine's processing industry into global value chains provides for the creation of
effective policies and institutions, aimed at eliminating restrictions in the the Ukrainian producers integration into
international production networks. Key words: international trade, trade policy, industry, global value chains, export potential, trade integration. JEL Classіfіcatіon E 23, F01, F13, F15, F62 Актуальність теми дослідження. Світ змінюється: поява нових технологій та їх стрімке поширення,
зростання обсягів світової торгівлі та інвестиційних потоків призводить до поглиблення економічної глобалізації та
розвитку гібридного виробництва. Глобальне переміщення виробничих мереж транснаціональними компаніями та
включення в ланцюги створення доданої вартості виробників країн з різним рівнем розвитку посилюють важливість
досліджень можливостей інтеграції промисловості України до глобальних виробничих мереж. Вбудовуючись в
ланцюги доданої вартості, промислові виробники України вимушені пристосовуватись до вимог світового попиту. Це призводить до змін у структурі промисловості: в тих секторах, які орієнтовані на експорт, зростання числа
виробників відбувається більш високими темпами, ніж в інших секторах. З іншого боку, подальша лібералізація
зовнішньої торгівлі призводить до посилення конкуренції на внутрішньому ринку у зв’язку зі зростаючим імпортом,
що є джерелом додаткових ризиків. У цих умовах зміна стратегічної поведінки виробників і, як наслідок, структури
промислового виробництва країни та окремих секторів, є одним з напрямків адаптації національного виробництва
до нових викликів і можливостей, обумовлених міжнародною торгівлею. OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS у
у
у
Досягнення поставленої мети передбачає виконання наступних завдань: - розкрити існуючі підходи до визначення поняття формування ланцюгів створення доданої вартості та
здійснити їх групування за сутнісними ознаками, а саме за масштабом і територіальним охопленням,
структурою управління і способам вимірювання доданої вартості; - розкрити існуючі підходи до визначення поняття формування ланцюгів створення доданої вартості та
здійснити їх групування за сутнісними ознаками, а саме за масштабом і територіальним охопленням,
структурою управління і способам вимірювання доданої вартості; ру
ур
у р
р
р
- на основі аналізу зрушень в розміщенні та управлінні глобальними ланцюгами вартості на рівні
галузей і регіонів світу сформулювати особливості та сучасні тренди розвитку глобальних ланцюгів вартості
в переробній промисловості; у
у
у
- на основі аналізу зрушень в розміщенні та управлінні глобальними ланцюгами вартості на рівні
галузей і регіонів світу сформулювати особливості та сучасні тренди розвитку глобальних ланцюгів вартості
в переробній промисловості; р р
р
- дослідити можливості та існуючі ризики при вбудовуванні в ланцюги створення д
країн, що розвиваються; и можливості та існуючі ризики при вбудовуванні в ланцюги створення доданої вартості дл
аються; - розробити практичні рекомендації щодо вибору найбільш ефективних способів інтеграції до
глобальних ланцюгів доданої вартості для переробної промисловості України як надійного постачальника
продукції з більш високим ступенем переробки. - розробити практичні рекомендації щодо вибору найбільш ефективних способів інтеграції до
глобальних ланцюгів доданої вартості для переробної промисловості України як надійного постачальника
продукції з більш високим ступенем переробки. Методи дослідження. У роботі використані загальнонаукові методи: абстрактно-логічний, індукції та
дедукції, системного підходу; аналізу і синтезу, а також спеціальні методи дослідження: аналіз можливостей і
обмежень, які постають при вбудовуванні окремого виробника в ланцюги доданої вартості; статистичних
порівнянь, групування, вибірки; структурно-функціонального аналізу, експертних оцінок при розробці
рекомендацій щодо можливих напрямів сприяння інтеграції переробної промисловості України до глобальних
ланцюгів доданої вартості. р
Постановка проблеми. Використання сприятливих можливостей та мінімізація загрозливих чинників
лібералізації зовнішньої торгівлі у все більш відкритому та конкурентному середовищі ставить нові вимоги
для виробничих підприємств. Розвиток сучасних технологій, в тому числі інформаційних, ініціює розвиток
глобальних виробничих систем та надає можливості для включення в глобальні ланцюжки доданої вартості
значної кількості країн, в яких є відповідні професійні компетенції, наявний кадровий потенціал та доступні
ресурси за конкурентною ціною. Це і є конкурентними перевагами нашої країни, що дозволяє українським
виробниками стати надійними учасниками глобальних ланцюгів доданої вартості. у
Результати дослідження. OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS Актуальність особливостей формування ланцюгів створення доданої вартості в переробній
промисловості з точки зору виробництва та торгівлі, можливостей залучення окремих сфер переробної
промисловості України до вищих ланок глобальних ланцюгів доданої вартості є надзвичайно гострою в
сучасних умовах посилення міжнародної конкуренції. у
у
у
Ступінь дослідження даної проблеми вченими. Різні аспекти розвитку глобальних ланцюгів доданої
вартості промисловості знайшли відображення у багатьох наукових працях зарубіжних та вітчизняних учених. Концептуальні засади досліджуваного поняття сформували зарубіжні науковці різних напрямів і шкіл сучасної
економічної думки. До їх числа можна віднести праці П.Марша, Р. Каплінського та Морріса, К.Шваба, Р.Райха,
Д. Родріка, Е.Райнерта, С.Вебера, П.Лабаста, Г.Джереффі та інших. Р.Райх наголошує на тому, що в сучасній
глобальній економіці інтернаціоналізація економіки перетворює стадії виробництва продукції на послідовні
ланки міжнародного кооперованого виробництва. К.Шваб в своїх дослідженнях зазначає, що характер
глобальних змін є настільки фундаментальним, що суспільству необхідно підготуватись до викликів часу як
великих можливостей, так і потенційних небезпек. В Україні різним аспектам інтегрованого промислового
розвитку та особливостям формування ланцюгів доданої вартості присвячують свої роботи такі науковці, як:
Геєць В.М., Вишневський О.С., Дейнеко Л.В., Кизим М.О., Кваша Т.К., Ляшенко В.І., Мусіна Л.А., Пятницький
В.Т., Сіденко В.Р. та ін. Разом з тим, в умовах посилення впливу інтеграційних процесів на розвиток
міжнародної торгівлі та виробництва залишається недостатньо дослідженою проблема вибору найбільш
ефективних способів інтеграції до глобальних ланцюгів доданої вартості для переробної промисловості
України як надійного постачальника продукції з більш високим ступенем переробки. р
д
р ду ц
у
р р
Предметом дослідження є теоретичні та методичні підходи щодо розвитку ланцюгів доданої вартості
в контексті промислового розвитку. Об'єктом дослідження є особливості формування ланцюгів створення доданої вартості та способи
інтеграції до них для переробної промисловості України як надійного постачальника продукції з більш високим
ступенем переробки. Об'єктом дослідження є особливості формування ланцюгів створення доданої вартості та способи
інтеграції до них для переробної промисловості України як надійного постачальника продукції з більш високим
ступенем переробки. Економічний вісник університету | Випуск № 39 67 ЕКОНОМІКА ТА УПРАВЛІННЯ Метою дослідження є розробка пропозицій щодо можливих напрямів сприяння інтеграції переробної
промисловості України до глобальних ланцюгів створення доданої вартості, що призведе до диверсифікації
промисловості та створення нових виробництв з більш глибоким ступенем переробки, що забезпечить
міжгалузеві структурні зміни і перехід економіки на новий технологічний рівень. Економічний вісник університету | Випуск № 39 OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS Сьогодні в світовій науковій думці не існує єдиного підходу щодо визначення
поняття ланцюгів доданої вартості, оскільки концепція їх формування використовується для різноманітних
цілей в залежності від результатів економічної та соціальної організації. В контексті посилення впливу
глобалізації, що спричинила трансформацію традиційних форм підприємництва до більш гнучких, мережевих
форм співробітництва, стрімкого розвитку набувають такі інтегровані структури, що характеризується
складними структурними та процесними зв’язками між всіма учасниками на принципах стратегічного
партнерства як ланцюги або мережі створення доданої вартості [1]. Характеризуючи механізм формування ланцюгів створення доданої вартості, різні дослідники
акцентують увагу на їх різних особливостях. Відомий фахівець в області «мережевої економіки» –
М. Кастельс, після проведення досліджень у США, Японії, Тайвані, Південній Кореї, Гонконгу, Китаї, Західній
Європі (Англії, Франції), Росії, зазначає, що в умовах ключової ролі технології генерування знань, обробки
інформації та символічної комунікації досягнення певного рівня продуктивності та існування конкуренції
можливе лише всередині глобальної взаємозалежної мережі [2, с. 81], а нові економічні форми будуються
навколо глобальних мережевих структур капіталу, управління та інформації, тобто компанії, фірми і, у все
більшій мірі, інші організації та інституції об’єднуються в мережі різної конфігурації, структура яких
характеризує відхід від традиційних відмінностей між великими корпораціями і малим бізнесом, охоплюючи
сектори та економічні групи, організовані за географічним принципом [3]. Відомий економіст П.Марш пояснює неминучість інтеграції підприємств в мережеві структури та
необхідність формування ефективних ланцюгів поставок в глобалізованій економіці [4, с. 11]. Лауреат Нобелівської премії з економіки Пол Кругман відзначає, що якщо на початку XX ст. той чи інший
споживчий товар міг бути експортований тільки один раз, то сьогодні його можна вивезти багато разів. Товар,
виготовлений в одній країні, може бути зібраний з компонентів, зроблених в інших країнах, а вони в свою чергу
можуть бути укомплектовані з субкомпонентів, виготовлених в інших країнах. В результаті інтегрована в
глобальне виробництво кінцевого продукту торгівля може включати в себе кілька вартостей, доданих на всіх
стадіях виробництва [5, с. 334]. Р. Каплінський та М. Моріс зазначають, що глобальні ланцюги доданої вартості (ГЛДВ) охоплюють всі
сектори економіки, мають широкий географічний розподіл та визначають спеціалізацію окремих фірм та країн
в цілому на окремих стадіях виробничого процесу. Враховуючи це, ГЛДВ включають в себе сукупність заходів
для розробки продукти або послуги, починаючи від його концепції через послідовні етапи виробництва
(включаючи комбінацію фізичних змін сировинних компонентів та внеску супутніх виробничих послуг),
завершуючи доставкою кінцевому споживачу та після продажні послуги [6, с. 4]. Р. Каплінський та М. OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS країн ЄС28 і Китаю, падіння світових цін н
товарів українського експорту; • активізація торгівельної економічного співробітництва України та її включення до глобальних
торгівельних потоків, що обумовлено імплементацією Угоди про асоціацію між Україною та ЄС, підписання
Угод про створення зони вільної торгівлі (ЗВТ) з іншими країнами (Канадою та ін.); р
р
р
(
)
р
(
)
• впровадження високотехнологічної стратегії підвищення конкурентоспроможнос
рамках четвертої промислової революції, відомої як «Індустрія 4.0»; острення боротьби за сировинні ресурси та ринки збуту продукції; • загострення боротьби за сировинні ресурси та ринки збуту продукції; • загострення боротьби за сировинні ресурси та ринки збуту продукції; р
р
р
р
ур
р
у у
р
у
• ескалація торговельного протекціонізму розвинених країн у вигляді застосування торговельних
інструментів: введення високих мит на експорт сировини та імпорт переробленої продукції, застосування
технічних бар'єрів, використання санітарних і фіто-санітарних норм, правил визначення країни походження
товарів та інших нетарифних заходів. р ф
д
упи внутрішніх чинників, пов’язаних з внутрішньо-політичною ситуацією можна віднести: р
р ф
До групи внутрішніх чинників, пов’язаних з внутрішньо-політичною ситуацією можна віднести: • руйнування промислового виробництва на територіях Донецької та Луганської областей, зокрема
металургійного та хімічного, продукція яких була чи не найвагомішою складовою експортних поставок з України; ур
, р ду ц
у
д
р
• блокада поставок ресурсів з територій АТО та припинення зовнішньоторговел
осійською Федерацією (РФ) яка тривалий час була домінуючим торговим партнером Украї р
у
у
р
р
поставок ресурсів з територій АТО та припинення зовнішньоторговельних відносин
рацією (РФ), яка тривалий час була домінуючим торговим партнером України. • блокада поставок ресурсів з територій АТО та припинення зовнішньоторговельних відносин з
Російською Федерацією (РФ), яка тривалий час була домінуючим торговим партнером України. • загальне
погіршення
фінансово-економічного
стану
вітчизняних
підприємств
і
скорочення • блокада поставок ресурсів з територій АТО та припинення зовнішньоторговельних відносин з
Російською Федерацією (РФ), яка тривалий час була домінуючим торговим партнером України. • загальне погіршення фінансово-економічного стану вітчизняних підприємств і скорочення
платоспроможності населення у 2016–2017 рр. д р ц
(
),
р
у
д
у
р
р
р
• загальне погіршення фінансово-економічного стану вітчизняних підприєм
платоспроможності населення у 2016–2017 рр. При цьому під впливом вище зазначених зовнішніх і внутрішніх факторів останнім часом вже відбулися
певні зрушення в товарній структурі експорту. На жаль, продовжує зростати частка сировини і продукції з
низьким рівнем переробки. Якщо у 2012 р. традиційні для України аграрна сировина і чорні метали становили
48,9% експорту, то в 2016 р. OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS Вбудовування в ГЛДВ дозволяє мінімізувати ключові ризики: Як зазначається в доповіді ЮНКТАД «Доповідь про торгівлю та розвиток за 2016 рік», підключення до
ГЛДВ є першим щаблем вгору по вертикалі індустріалізації та більш збалансованої участі в глобальному
торгівельному просторі Вбудовування в ГЛДВ дозволяє мінімізувати ключові ризики: Д
р
щ
ру
р
ду
р
ц
торгівельному просторі. Вбудовування в ГЛДВ дозволяє мінімізувати ключові ризики: у
у
у
у
1. При створенні повного циклу виробництва певного товару всередині країни, що вимагає значної
кількості ресурсів: інвестицій, технологій, кваліфікованих працівників, компетенцій; 1. При створенні повного циклу виробництва певного товару всередині країни, що вимагає значної
кількості ресурсів: інвестицій, технологій, кваліфікованих працівників, компетенцій; р
ур
ц ,
,
ф
р ц
,
ц ;
2. При самостійному виході на ринки з жорсткою конкуренцією, особливо для країн, що розвиваються. 2. При самостійному виході на ринки з жорсткою конкуренцією, особливо для країн, що розвиваються. Саме це наразі є надзвичайно актуальним для України, де переробна промисловість не має достатнього
рівня готовності до майбутніх викликів що несе певні загрози в умовах посилення глобальної конкуренції та р
у
д
р
р
ур
ц
,
д
р
, щ
р
Саме це наразі є надзвичайно актуальним для України, де переробна промисловість не має достатнього
рівня готовності до майбутніх викликів, що несе певні загрози в умовах посилення глобальної конкуренції та
не дозволить українським виробникам конкурувати зі світовими лідерами вже в середньостроковій
перспективі. Перед Україною сьогодні постали численні виклики, пов’язані з деіндустріалізацією
промисловості та необхідністю географічної й товарної диверсифікації експорту. Сучасні трансформації в
зовнішньоторговельній діяльності промисловості України обумовлені впливом групи зовнішніх і внутрішніх
чинників. Так, можна виділити зовнішні чинники, обумовлені викликами глобального ринку, а саме: у
• уповільнення динаміки розвитку світової економіки, у т.ч. країн ЄС28 і Китаю, пад
традиційні види товарів українського експорту; у
у
ення динаміки розвитку світової економіки, у т.ч. OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS Моріс зазначають, що глобальні ланцюги доданої вартості (ГЛДВ) охоплюють всі
сектори економіки, мають широкий географічний розподіл та визначають спеціалізацію окремих фірм та країн
в цілому на окремих стадіях виробничого процесу. Враховуючи це, ГЛДВ включають в себе сукупність заходів
для розробки продукти або послуги, починаючи від його концепції через послідовні етапи виробництва
(включаючи комбінацію фізичних змін сировинних компонентів та внеску супутніх виробничих послуг),
завершуючи доставкою кінцевому споживачу та після продажні послуги [6, с. 4]. р
у
у
у
р
у
[
]
Патрік Лоу виділяє сервісну діяльність (послуги) як невід’ємний компонент існуванн
вартості (ЛДВ) та виокремлює поняття «сервісної науки», що пояснює значення послуг в Патрік Лоу виділяє сервісну діяльність (послуги) як невід’ємний компонент існування ланцюгів доданої
вартості (ЛДВ) та виокремлює поняття «сервісної науки», що пояснює значення послуг в процесі отримання 68
Економічний вісник університету | Випуск № 39 68 ЕКОНОМІКА ТА УПРАВЛІННЯ цінності в ланцюгах поставок. Послуги в даному контексті поєднують виробництво, технології, підприємництво
та споживачів в інноваційному циклі конструктивної діяльності. Тобто, при формуванні ЛДВ найбільш вигідні
їх компоненти (в основному послуги: дослідження та розробки, маркетинг, центр прибутку) знаходяться
всередині об’єднання, а менш вигідні (виробництво та збірка) – поза об’єднанням [7, с. 76]. Такі підходи обумовлюють можливість використання терміну «ланцюг» для позначення системи
взаємовідносин в контексті управління створення доданої вартості продукту, оскільки вартість продукту
збільшується за рахунок різних видів діяльності, включаючи переробку, складання, пакування,
транспортування, брендінг, роздрібну торгівлю. р
р у
,
р
д
, р
др
у
р
Більшість дослідників ГЛДВ підкреслюють можливості, які виникають при вбудовуванні до них для країн,
що розвиваються. З часів появи ГЛДВ (середина 1980-х років) відбулись значні зміни в формах взаємодії
ланок ланцюгів, а саме перерозподіл технологічних стадій виробництва товарів та послуг між виробниками,
що знаходяться в різних країнах. Сьогодні це масштабні сітьові структури міжнародного виробничого та
науково-технічного кооперування, які охоплюють значну кількість ланок в географічно розрізнених країнах. й етап розвитку міжнародного поділу праці; р
у
р
у
р
• зростання обсягів міжнародної торгівлі, зокрема товарами проміжного споживання; • посилення впливу ТНК та фінансових ринків; • посилення впливу ТНК та фінансових ринків; • розвиток інтеграційних утворень та підписання нових міжнародних торгівельних угод • розвиток інформаційних технологій та поширення доступу до інформаційного простору;
• ріст концентрації ринків. р
р
р
Як зазначається в доповіді ЮНКТАД «Доповідь про торгівлю та розвиток за 2016 рік», підключення до
ГЛДВ є першим щаблем вгору по вертикалі індустріалізації та більш збалансованої участі в глобальному
торгівельному просторі. OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS їх частка зросла до 57,6 %, що підтверджує зберігання тенденції сировинної
орієнтації українського експорту та вимагає рішучих дій для реструктуризації експорту шляхом його
переорієнтації на продукцію з доданою вартістю. Саме тут і можна реалізувати позитивні можливості інтеграції в ГЛДВ. Цей процес уже розпочався в
Україні. Такі галузі як машинобудування (виробництво комплектуючих для автомобільної промисловості),
легка, меблева (виробництво сандвіч панелей для будівництва) та інші вже залучені в світові виробничі
мережі. Економічний вісник університету | Випуск № 39 69 ЕКОНОМІКА ТА УПРАВЛІННЯ Загальноприйнятим показником участі у ГЛДВ є торгівля проміжними товарами, яка починаючи з 2011
року має постійну тенденцію до зростання. Так, за даними ЮНІДО [8], її частка в структурі світової торгівлі
досягла половини та продовжує щорічно зростати. В структурі експорту країн «Великої двадцятки»,
насамперед Китаю, Індії, Японії та Кореї, 30-60% становить проміжна продукція, яка потім проходить
додаткову переробку в країнах-партнерах, торгівля якої проходить в межах ГЛВД. Тенденції експорту промислових проміжних товарів, які використовуються для подальшої переробки
свідчать про значний потенціал інтеграції української промисловості до інтеграції в ланцюги доданої вартості. Так, за даними Світового банку (порталу WITS) [9], частка експорту проміжної продукції України становить
44,2% у 2015 році, що повністю відповідає показникам активних учасників ГЛВД. Водночас, відбувається
скорочення питомої ваги експорту проміжної продукції в загальній структурі промислового експорту з 49,3% у
2010 до 44,2% у 2015 році. Ще одним чинником, що підтверджує важливість невідкладних дій зі стимулювання
інтеграції в ГЛВД української переробної промисловості, є зміни товарної складової міжнародної торгівлі, а
саме суттєве зростання експорту сировини (з 16,2% у 2010 році до 31,1% у 2015 році), що показано на рис.1. 70
Економічний вісник університету | Випуск № 39
Рисунок 1. Структура експорту промислової продукції України у 2010 та 2015 рр. Джерело: побудовано авторами за даними Світового банку [9]. Регіональний характер ГЛВД також посилює додаткові переваги, пов’язані з географічною диверсифікацією. Відбувається географічна переорієнтація інтеграції української промисловості до ГЛДВ: скорочення
співпраці з РФ майже вдвічі та нарощення обсягів реалізації промислової продукції проміжного споживання
до країн африкансько-азійського вектору (Індія, Єгипет, Китай) та європейських країн (Польща, Болгарія), що
показано на рис. 2. Рисунок 2. Експорт проміжних товарів України з 2010-2015 рр. Джерело: побудовано авторами за даними Світового банку [9]. Проміжні
товари
49,3%
Сировина
16,2%
Споживчі
товари
20,0%
Інвестиційні
товари…
2010
Проміжні
товари
44,2%
Сировина
31,1%
Споживчі
товари
16,0%
Інвестиційні
товари…
2015 Рисунок 1. Структура експорту промислової продукції України у 2010 та 2015 рр. Джерело: побудовано авторами за даними Світового банку [9]. ЕКОНОМІКА ТА УПРАВЛІННЯ Аналіз даних свідчить про те, що складний період падіння обсягів українського промислового експорту
(скорочення більш ніж вдвічі порівняно з 2012 р. – з 57,3 млрд дол. США до 27,5 млрд дол. США в 2016 р.)
змінюється на позитивну динаміку 2017 року. Аналіз даних свідчить про те, що складний період падіння обсягів українського промислового експорту
(скорочення більш ніж вдвічі порівняно з 2012 р. – з 57,3 млрд дол. США до 27,5 млрд дол. США в 2016 р.)
змінюється на позитивну динаміку 2017 року. у д
у
р
у
Сьогодні відбувається покращення ситуації: у 2017 р. спостерігаємо зростання промислового експорту
на 5,5 млрд дол. США, що становить 120,1% до 2016 р (рис. 3). у
у
р
у
Сьогодні відбувається покращення ситуації: у 2017 р. спостерігаємо зростання промислового експорту
на 5,5 млрд дол. США, що становить 120,1% до 2016 р (рис. 3). Рисунок 3. Динаміка експорту та імпорту промислової продукції за 2013-2017 рр., у % до попереднього року
Джерело: побудовано авторами за даними Держстату України [10]. 90,6
85,0
66,5
93,6
120,1
90,3
71,7
69,9
104,4
127,2
0
20
40
60
80
100
120
140
2013
2014
2015
2016
2017
%
Експорт
Імпорт Рисунок 3. Динаміка експорту та імпорту промислової продукції за 2013-2017 рр., у % до
Джерело: побудовано авторами за даними Держстату України [10]. Незважаючи на це, рівень відкритості української економіки залишається високим, що зумовлює її
вразливість до зовнішніх шоків, що передбачає інтегрованість внутрішнього виробництва до світового, а отже,
зовнішньоторговельну відкритість національної промисловості. Ступінь відкритості можна визначити за
показником експортної та імпортної квоти, яка для України за 2017 рік є в межах 30% та підтверджує
посилення інтеграції України в світовий торгівельно-економічний простір (рис. 4). Це означає, що в коротко- і середньостроковій перспективі Україна зможе отримати більше доходів за
рахунок збільшення виробництва у відносно вузькому діапазоні традиційних товарів, що експортуються. В довгостроковій перспективі товарна й географічна диверсифікація експорту могла б зміцнити економічну
стійкість української промисловості та прискорити процес передачі нових технологій виробництва. Рисунок 4. Графік відкритості економіки за квотами
Джерело: побудовано авторами за даними Держстату України [10]. 28,3
33,5
32,4
29,4
29,4
38,9
38,8
39,5
40,0
42,3
0
5
10
15
20
25
30
35
40
45
2013
2014
2015
2016
2017
%
Експортна квота
Імпортна квота Рисунок 4. Графік відкритості економіки за квотами
Джерело: побудовано авторами за даними Держстату України [10]. Рисунок 4. Графік відкритості економіки за квотами
Джерело: побудовано авторами за даними Держстату України [10]. OPPORTUNITIES FOR THE INTEGRATION
OF THE UKRAINIAN INDUSTRY INTO GLOBAL VALUE CHAINS Проміжні
товари
49,3%
Сировина
16,2%
Споживчі
товари
20,0%
Інвестиційні
товари…
2010
Проміжні
товари
44,2%
Сировина
31,1%
Споживчі
товари
16,0%
Інвестиційні
товари…
2015 Проміжні
товари
44,2%
Сировина
31,1%
Споживчі
товари
16,0%
Інвестиційні
товари…
2015 Інвестиційні
товари… Рисунок 1. Структура експорту промислової продукції України у 2010 та 2015 рр. Джерело: побудовано авторами за даними Світового банку [9]. Регіональний характер ГЛВД також посилює додаткові переваги, пов’язані з географічною диверсифікацією. Відбувається географічна переорієнтація інтеграції української промисловості до ГЛДВ: скорочення
співпраці з РФ майже вдвічі та нарощення обсягів реалізації промислової продукції проміжного споживання
до країн африкансько-азійського вектору (Індія, Єгипет, Китай) та європейських країн (Польща, Болгарія), що
показано на рис. 2. р
р
Д
д д
р
,
р ф
д
р
ф
ц
Відбувається географічна переорієнтація інтеграції української промисловості до ГЛДВ: скорочення
співпраці з РФ майже вдвічі та нарощення обсягів реалізації промислової продукції проміжного споживання
до країн африкансько-азійського вектору (Індія, Єгипет, Китай) та європейських країн (Польща, Болгарія), що
показано на рис. 2. Відбувається географічна переорієнтація інтеграції української промисловості до ГЛДВ: скорочення
співпраці з РФ майже вдвічі та нарощення обсягів реалізації промислової продукції проміжного споживання
до країн африкансько-азійського вектору (Індія, Єгипет, Китай) та європейських країн (Польща, Болгарія), що
показано на рис. 2. Рисунок 2. Експорт проміжних товарів України з 2010-2015 рр. Джерело: побудовано авторами за даними Світового банку [9]. Рисунок 2. Експорт проміжних товарів України з 2010-2015 рр. Джерело: побудовано авторами за даними Світового банку [9]. Рисунок 2. Експорт проміжних товарів України з 2010-2015 рр. Джерело: побудовано авторами за даними Світового банку [9]. Рисунок 2. Експорт проміжних товарів України з 2010-2015 рр. Джерело: побудовано авторами за даними Світового банку [9]. Економічний вісник університету | Випуск № 39 Економічний вісник університету | Випуск № 39 70 ЕКОНОМІКА ТА УПРАВЛІННЯ У той же час міг сприйняття української економіки як відкритою дещо знижується, оскільки це сприйняття
в основному ґрунтується на функціональної відкритості (фактично залучення країни до різних форм
міжнародного економічного співробітництва). Вона не враховує так звану «інституційну відкритість», а саме
обмеження торгівлі та мобільності капіталу (імпортні бар'єри, тарифні ставки, мито тощо). Участь українських
компаній в глобальних ланцюгах доданої вартості залишається не дуже активною. Це пов'язано, в першу
чергу, з низькою конкурентоспроможністю вітчизняних компаній. Іншою причиною є тривала адаптація Економічний вісник університету | Випуск № 39 71 ЕКОНОМІКА ТА УПРАВЛІННЯ національних стандартів в галузі сертифікації якості. Іноземні компанії не купують товари від виробників, які
не можуть гарантувати високий рівень якості продукту та походження сировини. національних стандартів в галузі сертифікації якості. Іноземні компанії не купують товари від виробників, які
не можуть гарантувати високий рівень якості продукту та походження сировини. Перспективами більш активної участі України в глобальних ланцюгах доданої вартості, зокрема в сфері
літакобудування, пов'язані з реалізацією конкурентних переваг у вантажних перевезеннях та розширенні
регіональних та місцевих рейсів. Обидва ніші все ще слабо розвинені в порівнянні з зовнішніми ринками, хоча
вони поступово розширюються. у
р
р
Особливістю ГЛДВ є зниження бар’єрів на вході в нижні щаблі ланцюжка створення доданої вартості,
що спрощує Україні інтеграцію в глобальний експорт готового продукту. Проте, ці умови спрощення доступу
на перші ланки також можуть виступати як гальмуючий чинник для модернізації, оскільки обмежений
інституційний розвиток, недостатнє технологічне забезпечення виробництва, слабкий взаємозв’язок з
внутрішньою економікою призводить до спеціалізації на первинній переробці сировині. До того ж, слабка
торгівельна політика разом з несприятливим зовнішньоекономічним іміджем країни можуть призвести до
неглибокої інтеграції на низьких ланках ГЛДВ. В цьому сенсі вирішальну роль відіграє характер участі в ГЛДВ. Країни, що можуть розвивати виробничі
потужності відповідно до потреб міжнародних виробничих мереж та можуть зайняти потенційно високе місце
в світовому розподілі виробничих задач, повинні мати потужні можливості для економічного зростання, а саме
активність іноземного інвестування, доступ до ресурсів. Натомість в Україні ці можливості істотно обмежені. в світовому розподілі виробничих задач, повинні мати потужні можливості для економічного зростання, а саме
активність іноземного інвестування, доступ до ресурсів. Натомість в Україні ці можливості істотно обмежені. Проте, існують й певні ризики. Так, участь в ГЛДВ в умовах низької динаміки експорту готової продукції,
може призвести до гальмування структурної трансформації промисловості в частині зростання поставок
товарів первинної переробки. Як це відбулось в країнах Латинської Америки. Проте, існують й певні ризики. Економічний вісник університету | Випуск № 39 ЕКОНОМІКА ТА УПРАВЛІННЯ Так, участь в ГЛДВ в умовах низької динаміки експорту готової продукції,
може призвести до гальмування структурної трансформації промисловості в частині зростання поставок
товарів первинної переробки. Як це відбулось в країнах Латинської Америки. Крім того ГЛДВ можуть призвести до нерівності в доходах розвинутих країн та країн, що розвиваються. Факторний аналіз створення вартості показує, що зростання питомої ваги капіталу й оплати праці
висококваліфікованих робітників знаходить віддзеркалення у зменшенні доходів робітників середньої та
низької кваліфікації. Так, згідно даних Всесвітньої бази «Витрати-випуск» в 1995 р. на оплату
висококваліфікованих працівників припадало 55% вартості готових виробів, а в 2008 р. вже 63%. Це вимагає розробки і впровадження ефективних заходів державного менеджменту з підтримки української
промисловості в умовах дії сучасних торговельних угод, що посилює актуальність формування та реалізації
ефективної торговельної політики України. До того ж враховуючи експортозалежність економіки України (в
структурі ВВП частка експортно-імпортних операцій коливається у межах 45-60%), відсутність довгострокової
стратегії зовнішньоекономічної політики в Україні призводить до неможливості захисту національного
виробництва, використовуючи повною мірою ті механізми, які успішно застосовують інші країни світу. р
р
у
р
у
у
р
у
Враховуючи зміну географічних та товарних векторів вітчизняного експорту, подальші перспективи
участі України в глобальному торгівельному просторі залежатимуть від того, чи буде досягнуто зростання
конкурентоспроможності внутрішньої частини ланцюга доданої вартості. Впровадження ефективних
інструментів стимулювання входження переробної промисловості України до глобальних ланцюгів доданої
вартості передбачає створення ефективної політики та інституцій, націлених на усунення обмежень при
інтеграції українського виробника до міжнародних виробничих мереж. З огляду на зміни державної підтримки і відхід від прямого субсидування окремих галузей, що
унеможливлює використання методів прямої державної підтримки (експортне субсидіювання), першочерговим
завданням є створення ефективного конкурентного середовища та позитивного бізнес-клімату, що включатиме
наступні напрями, які стимулюють виробництво і торгівлю на окремих ланках ланцюгів доданої вартості: 1. Виробництво сировини та первинних продуктів:
•
гармонізації вітчизняних норм в сфері стандартизації та сертифікації сировини та матеріалів,
що передбачатиме вдосконалення адміністративно-правового регулювання в галузі стандартизації, якості
продукції, метрології і сертифікації, яке здійснюється в державі, що націлене на скорочення випуску неякісної,
а в окремих випадках і небезпечної продукції, що виготовляється з порушенням правил, норм і стандартів,
прийнятих в Україні та світі; оновлення системи технічного регулювання; усунення дублювання у сфері
державного нагляду за відповідністю продукції; спрощення процедур походження українських матеріалів;
розширення участі інституцій технічного регулювання у Європейських та міжнародних форумах та заходах,
забезпечуючи координацію та ефективну системи обміну інформацією. ф
у
у
ф р
ий сегмент ланцюга доданої вартості (обробка, збирання та виготовлення продуктів
м додавання вартості): 2. Список використаних джерел 1. Steve New. McDonald’s and the Challenges of a Modern Supply Chain. Harvard Business Review, 2015. URL: https://hbr.org/2015/02/mcdonalds-and-the-challenges-of-a-modern-supply-chain 1. Steve New. McDonald’s and the Challenges of a Modern Supply Chain. Harvard Business Review, 2015. URL: https://hbr.org/2015/02/mcdonalds-and-the-challenges-of-a-modern-supply-chain
2. Кастельс М. Информационная эпоха: экономика, общество и культура: Монография. Пер. с англ. О. И. Шкаратана. Москва:
Высш. шк. Экономики,
2000. 606
с. URL:
http://www.lavkamirov.com/cyberpunk/books/nonfiction_books.html 2. Кастельс М. Информационная эпоха: экономика, общество и культура: Монография. Пер. с англ. О. И. Шкаратана. Москва:
Высш. шк. Экономики,
2000. 606
с. URL:
http://www.lavkamirov.com/cyberpunk/books/nonfiction_books.html p
y
p
_
3. Новая постиндустриальная волна на Западе. Антология. /Под редакцией В. Л. Иноземцева. Москва: Academia, 1999. 640 стр. 3. Новая постиндустриальная волна на Западе. Антология. /Под редакцией В. Л. Иноземцева. Москва: Academia, 1999. 640 стр. ,
р
4. Марш П. Новая промышленная революция. Потребители, глобализация и конец массового
производства / пер. с англ. А. Шоломицкой. Москва: Изд-во Института Гайдара, 2015. 420 с. 4. Марш П. Новая промышленная революция. Потребители, глобализация и конец массового
производства / пер. с англ. А. Шоломицкой. Москва: Изд-во Института Гайдара, 2015. 420 с. р
р
у
р
5. Krugman P. Growing World Trade: Causes and Consequences. Washington: DC: Brookings Papers on
Economic Activity, 1995. Vol. 1. P. 327-377. 5. Krugman P. Growing World Trade: Causes and Consequences. Washington: DC: Brookings Papers on
Economic Activity, 1995. Vol. 1. P. 327-377. y
6. Kaplinsky R., Morris M. A handbook for Value Chain Research / The International Development Research
Centre, 2001. 109 p. URL: http://www.prism.uct.ac.za/Papers/VchNov01.pdf 6. Kaplinsky R., Morris M. A handbook for Value Chain Research / The International Development Research
Centre, 2001. 109 p. URL: http://www.prism.uct.ac.za/Papers/VchNov01.pdf p
p
p
p
ue chains in a changing world Edited by Deborah K. Elms and Patrick Low. URL:
english/res_e/booksp_e/aid4tradeglobalvalue13_e.pdf 7. Global value chains in a changing world Edited by Deborah K. Elms a
https://www.wto.org/english/res_e/booksp_e/aid4tradeglobalvalue13_e.pdf p
g
g
p
g
p
8. Глобальні ланцюги доданої вартості та розвиток. Підтримка всеохоплюючого та сталого
промислового розвитку з боку ЮНІДО. Київ: ЮНІДО, 2015. 68 с. p
g
g
p
g
p
8. Глобальні ланцюги доданої вартості та розвиток. Підтримка всеохоплюючого та сталого
промислового розвитку з боку ЮНІДО. Київ: ЮНІДО, 2015. 68 с. 9. World Integrated Trade Solution. URL: http://wits.worldbank.org/
10. Державна служба статистики України. URL: http://www.ukrstat.gov.ua/ 9. World Integrated Trade Solution. URL: http://wits.worldbank.org/
10. Державна служба статистики України. URL: http://www.ukrstat.gov.ua/ ЕКОНОМІКА ТА УПРАВЛІННЯ Виробничий сегмент ланцюга доданої вартості (обробка, збирання та вигот
із меншим ступенем додавання вартості): •
адаптація промислових виробників до екологічних вимог задля інтеграції в глобальні ланцюги
доданої вартості: застосування технологій збереження води та енергії, зменшення використання ресурсів,
скорочення викидів парникових газів та забруднення навколишнього середовища, використання екологічно
чистих матеріалів та сировини; дотримання вимог екологічного маркування, стимулювання вітчизняних
підприємств для зменшення енергоємності виробництва та освоєння технологій нового виробництва,
зокрема, для розвитку альтернативної енергетики, збереження біорізноманіття тощо. • зростання купівельної спроможності покупців на кінцевих споживчих ринках, що передбачає
стимулювання розвитку внутрішнього ринку, а саме страхування підприємницьких ризиків у виробничій і
торговельній діяльності, державні закупівлі тощо; • зростання купівельної спроможності покупців на кінцевих споживчих ринках, що передбачає
стимулювання розвитку внутрішнього ринку, а саме страхування підприємницьких ризиків у виробничій і
торговельній діяльності, державні закупівлі тощо; 72
Економічний вісник університету | Випуск № 39 72 ЕКОНОМІКА ТА УПРАВЛІННЯ ЕКОНОМІКА ТА УПРАВЛІННЯ • розвиток системи логістичного обслуговування міжнародних товаропотоків, що передбачає
уніфікацію національних законодавств, гармонізацію транспортно-логістичної інфраструктури з тим, щоб вона
мала єдині технічні параметри і забезпечувала застосування єдиної технології доставки товарів у
міжнародному сполученні. Окремим стратегічним напрямом інтеграції України в європейську логістичну
систему є приведення транспортних засобів вітчизняних перевізників у відповідність до екологічних і технічних
стандартів ЄС; д р
;
• посилення спроможності ланцюга реагувати на кінцеві потреби споживачів та забезпечення
відкритості інформації про якість продукції; р
• посилення спроможності ланцюга реагувати на кінцеві потреби споживачів та забезпечення
відкритості інформації про якість продукції; р
ф р
р
р
у
• брендінг національного виробництва, посилення іміджу і репутації українського виробництва
певних продуктів шляхом маркетингової та дипломатичної діяльності зі стратегічного просування країни в
глобальній економічній системі, формування та використання конкурентних переваг на глобальному ринку з
метою створення іміджу та управління репутацією, щоб якнайповніше реалізувати інтереси держави. у
• брендінг національного виробництва, посилення іміджу і репутації українського виробництва
певних продуктів шляхом маркетингової та дипломатичної діяльності зі стратегічного просування країни в
глобальній економічній системі, формування та використання конкурентних переваг на глобальному ринку з
метою створення іміджу та управління репутацією, щоб якнайповніше реалізувати інтереси держави. Висновки. За результатами проведеного дослідження було здійснено оцінку інтеграції українських
виробничих компаній в міжнародні ланцюги доданої вартості. Зокрема, було визначено, що обсяги експорту
промислових проміжних товарів, які використовуються для подальшої переробки, свідчать про значний
потенціал інтеграції української промисловості до міжнародних ланцюгів доданої вартості – експорт проміжних
товарів, які використовуються для виготовлення кінцевого продукту за межами України, є найбільшим
(близько 44%) в структурі експорту України у 2016 році. До того ж, відбувається географічна переорієнтація
інтеграції української промисловості до глобальних виробничих мереж: скорочення співпраці з РФ майже
вдвічі та нарощення обсягів експорту промислової продукції проміжного споживання до європейських країн
(близько 66%), що робить українських експортерів частиною європейських виробничих ланцюжків. При цьому
важливим є включення українських виробничих компаній, які можуть розвивати виробничі потужності
відповідно до потреб міжнародних виробничих мереж та можуть зайняти потенційно високе місце в світовому
розподілі виробничих переділів, що дасть змогу досягти таких важливих цілей як зростання продуктивності
праці, розвиток нових технологій, диверсифікація експорту, збільшення можливостей для працевлаштування,
забезпечення більш високого рівня життя населення. Це вимагає більш стратегічної співпраці між урядом та
приватним сектором, зміцнення інституційної спроможності та використання інструментів торгівельної
політики для максимального збільшення доданої вартості на національному та глобальному рівнях. p
References 1. Steve New. (2015). McDonald’s and the Challenges of a Modern Supply Chain. Harvard Business Review. URL: https://hbr.org/2015/02/mcdonalds-and-the-challenges-of-a-modern-supply-chain. [in English]. 1. Steve New. (2015). McDonald’s and the Challenges of a Modern Supply Chain. Harvard Business Review. URL: https://hbr org/2015/02/mcdonalds-and-the-challenges-of-a-modern-supply-chain [in English] 2. Castells, Manuel (2000). Informatsionnaya epokha: ekonomika, obshchestvo i kul'tura: Monografiya. [Information Age: Economy, Society and Culture] Per. s angl. O. I. Shkaratana. Moskva: Vyssh. shk. Ekonomiki,
URL: http://www.lavkamirov.com/cyberpunk/books/nonfiction_books.html [in Russian] 2. Castells, Manuel (2000). Informatsionnaya epokha: ekonomika, obshchestvo i kul'tura: Monografiya. [Information Age: Economy, Society and Culture] Per. s angl. O. I. Shkaratana. Moskva: Vyssh. shk. Ekonomiki,
URL: http://www.lavkamirov.com/cyberpunk/books/nonfiction_books.html [in Russian] p
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3. Novaya postindustrial'naya volna na Zapade. Antologiya. [The new post-industrial wav
ology.]. Pod redaktsiey V. L. Inozemtseva. Moskva : Academia, 1999. 640 str [in Russian] 3. Novaya postindustrial naya volna na Zapade. Antologiya. [The new post-industrial wave in the West. Anthology.]. Pod redaktsiey V. L. Inozemtseva. Moskva : Academia, 1999. 640 str [in Russian]
4. Marsh, Peter. (2015). Novaya promyshlennaya revolyutsiya. Potrebiteli, globalizatsiya i konets massovogo
proizvodstva [The New Industrial Revolution: Consumers, Globalization and the End of Mass Production]. Per. s
angl. A. Sholomitskoy. Moskva : Izd-vo Instituta Gaydara, 420 s. [in Russian] 4. Marsh, Peter. (2015). Novaya promyshlennaya revolyutsiya. Potrebiteli, globalizatsiya i konets massovogo
proizvodstva [The New Industrial Revolution: Consumers, Globalization and the End of Mass Production]. Per. s
angl. A. Sholomitskoy. Moskva : Izd-vo Instituta Gaydara, 420 s. [in Russian] g
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5. Krugman, P. (1995) Growing World Trade: Causes and Consequences. Washington: DC: Brookings
Papers on Economic Activity. Vol. 1. P. 327-377. [in English]. Економічний вісник університету | Випуск № 39 73 Економічний вісник університету | Випуск № 39 ЕКОНОМІКА ТА УПРАВЛІННЯ 6. Kaplinsky, R. & Morris, M. (2001). A handbook for Value Chain Research / The International Development
Research Centre. 109 p. URL: http://www.prism.uct.ac.za/Papers/VchNov01.pdf [in English]. p
p
p
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g
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7. Global value chains in a changing world Edited by Deborah K. Elms and Patrick Low. URL:
https://www.wto.org/english/res_e/booksp_e/aid4tradeglobalvalue13_e.pdf [in English]. p
g
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8. Hlobalni lantsiuhy dodanoi vartosti ta rozvytok. Pidtrymka vseokhopliuiuchoho ta staloho promyslovoho
rozvytku z boku YuNIDO. [Global Value Chains and development. UNIDO’s support towards inclusive and
sustainable industrial development]. Kyiv: YuNIDO, 2015. 68 s [in Ukrainian]. 8. Hlobalni lantsiuhy dodanoi vartosti ta rozvytok. Pidtrymka vseokhopliuiuchoho ta staloho promyslovoho
rozvytku z boku YuNIDO. [Global Value Chains and development. UNIDO’s support towards inclusive and
sustainable industrial development]. Kyiv: YuNIDO, 2015. 68 s [in Ukrainian]. p
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9. World Integrated Trade Solution. URL: http://wits.worldbank.org/ [in English]. 10. Derzhavna sluzhba statystyky Ukrainy [State Statistics Service of Ukraine]. URL: http://www.ukrstat.gov.ua/
[in Ukrainian]. g
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g
]
10. Derzhavna sluzhba statystyky Ukrainy [State Statistics Service of Ukraine]. URL: http://www.ukrstat.gov.ua/
[in Ukrainian]. ДАНI ПРО АВТОРІВ у
р
,
д д
у , д ц
,
р
у
р
відділу промислової політики
ДУ «Інститут економіки та прогнозування НАН України»
e-mail: kyshnoksana@gmail.com
orcid.org/0000-0002-3853-584X
Researcher ID: T-1063-2017
Зарудна Ольга Станіславівна, молодший науковий співробітник відділу промислової політики
ДУ «Інститут економіки та прогнозування НАН України»
e-mail: olgaszua@gmail.com
orcid.org/0000-0002-7253-8091
Researcher ID: S-5188-2017 у
р
,
д д
у , д ц
,
р
у
р
відділу промислової політики
ДУ «Інститут економіки та прогнозування НАН України»
e-mail: kyshnoksana@gmail.com
orcid.org/0000-0002-3853-584X
Researcher ID: T-1063-2017
Зарудна Ольга Станіславівна, молодший науковий співробітник відділу промислової політики
ДУ «Інститут економіки та прогнозування НАН України»
e-mail: olgaszua@gmail.com
orcid.org/0000-0002-7253-8091
Researcher ID: S-5188-2017 відділу промислової політики g
Researcher ID: S-5188-2017 УДК 658.155 Халатур С. М.,
Пліско І. Г. Предмет дослідження – концептуальні, теоретико-методологічні й науково-практичні засади
управління прибутком на підприємствах. лідження – дослідити та описати механізм, чинники, резерви та шляхи підвищення рівн
ті підприємств національної економіки України в умовах фінансової глобалізації. прибутковості підприємств національної економіки України в умовах фінансової глобалізації. Методи дослідження. У статті застосовано сукупність методів наукового дослідження:
статистичний, історичний, нормативний, аналітичний. З методологічної точки зору, відзначимо, що
даний аналіз посилається на період 2010-2017 років. Результати роботи. Провівши дослідження шляхів підвищення прибутковості підприємств в умовах
ринку, можна зробити висновок, що через відсутність у більшості підприємств національної економіки
України ефективної бізнес-моделі підвищення прибутковості підприємства, існує загроза недоотримання
прибутків або взагалі отримання збитків. Стверджується, що підприємствам необхідно сформувати
оптимальну бізнес-модель підвищення прибутковості перед виконанням фінансово-господрської діяльності
для того, щоб досягти бажаних результатів. Проблема полягає в тому, що багато підприємств
використовують концепцію бізнес-моделі та бізнес-стратегії як взаємозамінні, що не є правильним. © Халатур С. М., Пліско І. Г., 2018 4
Економічний вісник університету | Випуск № 39 74
|
https://openalex.org/W4210347006
|
http://ejurnal.undana.ac.id/index.php/CMJ/article/download/5983/3315
|
Indonesian
| null |
Hubungan Antara Pengetahuan Dan Sikap Terhadap Perilaku Pencegahan Covid-19 Pada Mahasiswa Papua Dan Papua Barat Di Kupang
|
Cendana Medical Journal
| 2,021
|
cc-by
| 3,351
|
ABSTRAK Coronavirus (CoV) adalah keluarga besar virus yang menyebabkan penyakit mulai dari gejala
ringan sampai berat. Coronavirus Disease 2019 (COVID-19) adalah penyakit menular yang
disebabkan oleh Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Tanda
dan gejala umum infeksi COVID-19 antara lain gejala gangguan pernapasan akut seperti
demam, batuk dan sesak napas. Tujuan penelitian ini untuk menganalisis hubungan antara
pengetahuan dan sikap terhadap perilaku pencegahan COVID-19 pada mahasiswa Papua dan
Papua Barat di Kupang. Metode penelitian ini adalah analitik korelasi dengan pendekatan
cross sectional dan menggunakan uji Gamma. Pemilihan sampel menggunakan total
sampling. Hasil analisis statistik dengan Gamma didapatkan bahwa tidak ada hubungan yang
bermakna antara pengetahuan tentang COVID-19 terhadap perilaku pencegahan COVID-19
pada mahasiswa Papua dan Papua Barat di Kupang dengan nilai p=0.078. Hasil analisis
dengan Gamma didapatkan bahwa ada hubungan yang bermakna antara sikap tentang
COVID-19 terhadap perilaku pencegahan COVID-19 pada mahasiswa Papua dan Papua
Barat di Kupang dengan nilai p=0.000. Kesimpulan dari penelitian ini adalah tidak ada
hubungan yang bermakna antara pengetahuan tentang COVID-19 terhadap perilaku
pencegahan COVID-19 pada mahasiswa Papua dan Papua Barat di Kupang. Ada hubungan
yang bermakna antara sikap tentang COVID-19 terhadap perilaku pencegahan COVID-19
pada mahasiswa Papua dan Papua Barat di Kupang. Kata Kunci : COVID-19, Pengetahuan, Sikap, Perilaku, Pencegahan Kata Kunci : COVID-19, Pengetahuan, Sikap, Perilaku, Pencegahan Pada tanggal 31 Desember 2019,
World Health Organization (WHO) China
Country
Office
melaporkan
kasus
pneumonia yang tidak diketahui etiologinya
di Kota Wuhan, Provinsi Hubei, China. Pada tanggal 7 Januari 2020, China
mengidentifikasi kasus tersebut sebagai
jenis baru coronavirus. Pada tanggal 30
Januari 2020 WHO menetapkan kejadian
tersebut sebagai Kedaruratan Kesehatan
Masyarakat
yang
Meresahkan
Dunia
(KKMMD)/Public Health Emergency of
International Concern (PHEIC) dan pada
tanggal 11 Maret 2020, WHO sudah
menetapkan sebagai pandemi.(1) dapat menimbulkan gejala berat seperti
Middle
East
Respiratory
Syndrome
(MERS) dan Severe Acute Respiratory
Syndrome (SARS). dapat menimbulkan gejala berat seperti
Middle
East
Respiratory
Syndrome
(MERS) dan Severe Acute Respiratory
Syndrome (SARS). Coronavirus Disease 2019 (COVID-
19)
adalah
penyakit
menular
yang
disebabkan oleh Severe Acute Respiratory
Syndrome Coronavirus 2 (SARS-CoV-2). Tanda dan gejala umum infeksi COVID-19
antara lain gejala gangguan pernapasan
akut seperti demam, batuk dan sesak
napas.(2) Berdasarkan data WHO
secara
global per tanggal 26 Oktober 2020,
ada 42.745.212 kasus COVID 19
yang dikonfirmasi, termasuk 1.150.961kem
atian. Berdasarkan data WHO di Indonesi
a per tangal 26 Oktober 2020,ada 389.712 Coronavirus (CoV) adalah keluarga
besar virus yang menyebabkan penyakit
mulai dari gejala ringan sampai berat. Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Hubungan Antara Pengetahuan HUBUNGAN ANTARA PENGETAHUAN DAN SIKAP TERHADAP
PERILAKU PENCEGAHAN COVID-19 PADA MAHASISWA PAPUA
DAN PAPUA BARAT DI KUPANG Basterlita Y Rumere, I Nyoman Sasputra, Kartini Lidia, I Made Artawan ABSTRAK Ada
setidaknya dua jenis coronavirus yang
diketahui menyebabkan penyakit yang Universitas Nusa Cendana 298 Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Hubungan Antara Pengetahuan Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 kasus COVID−19
yang
dikonfirmasi
dengan 13.299 kematian.(3) diatas 50 tahun yang dapat terinfeksi
COVID-19.(6) Berdasarkan
data
Kementerian
Kesehatan (KEMENKES) di Nusa Teng
gara Timur per tanggal 26 Oktober 2020,
ada
652
kasus
COVID-19
yang
dikonfirmasi
dengan
7
kematian. Sedangkan wilayah di Nusa Tenggara
Timur dengan Transmisi Lokal terjadi di
Kota Kupang, Kab. Sumba Timur, Kab. Ende, dan Kab. Manggarai Barat.(4) Penelitian
sebelumnya
yang
dilakukan oleh Jesica Moudy dkk (2020)
tentang
Pengetahuan
terkait
Usaha
Pencegahan Coronavirus Disease 2019
(COVID-19) di Indonesia. Berdasarkan
hasil penelitian terdapat hubungan antara
pengetahuan dengan sikap dan pengetahuan
dengan tindakan individu. Penelitian
sebelumnya
yang
dilakukan oleh Sukesih, dkk (2020) tentang
Pengetahuan
dan
Sikap
Mahasiswa
Kesehatan tentang Pencegahan COVID-19
di Indonesia. Berdasarkan hasil penelitian
terdapat hubungan antara pengetahuan dan
sikap mahasiswa kesehatan. Saat ini, penyebaran SARS-CoV-2
dari manusia ke manusia menjadi sumber
transmisi
utama
sehingga
penyebaran
menjadi lebih agresif. Transmisi SARS-
CoV-2 dari pasien simptomatik terjadi
melalui droplet yang keluar saat batuk atau
bersin. Beberapa
laporan
kasus
menunjukkan dugaan penularan dari karier
asimtomatis, namun mekanisme pastinya
belum
diketahui. Kasus-kasus
terkait
transmisi dari karier asimtomatis umumnya
memiliki riwayat kontak erat dengan pasien
COVID-19.(5) Berdasarkan latar belakang diatas,
maka peneliti tertarik untuk mengadakan
penelitian
tentang
“Hubungan
antara
Pengetahuan dan Sikap terhadap Perilaku
Pencegahan COVID-19 pada Mahasiswa
Papua dan Papua Barat di Kupang”
sehingga
dari
hasil
penelitian
dapat
bermanfaat dan dapat dijadikan referensi
serta tambahan informasi dan pengetahuan. Menurut Kementerian
Komunikasi
dan Informatika (KEMKOMINFO) hasil
monitoring lalu lintas percakapan media
sosial berkaitan dengan virus corona
cenderung meningkat setiap harinya. Hoaks
dan disinformasi yang beredar beragam. Beragam informasi tentang COVID-19, ada
yang benar, ada yang salah, dan ada yang
salah METODE PENELITIAN Penelitian ini menggunakan desain
penelitian analitikal observasional dengan
rancangan
cross
sectional. Lokasi
penelitian
di
Kupang. Besar
sampel
penelitian ini adalah 74 orang, yang terdiri
dari mahasiswa program beasiswa afirmasi
pendidikan tinggi papua dan papua barat,
mahasiswa program beasiswa aku cinta
papua dan mahasiswa non beasiswa. Teknik sampling yang digunakan dalam
penelitian
ini
adalah
total
sampling. Analisis yang digunakan dalam penelitian
ini adalah analisis univariat dan bivariat. Analisis
univariat
dilakukan
dengan
mendeskripsikan masing-masing variable. Analisis bivariat untuk melihat hubungan
antar variabel yakni variabel bebas dan
variabel
terikat,
yaitu
mengetahui
hubungan antara pengetahuan dan sikap dipersepsikan oleh masyarakat. Peng
etahuan yang diperoleh dari sumber yang
tidak valid ini mempengaruhi perilaku
masyarakat dalam menghadapi situasi
pandemi COVID19.(6) Berdasarkan
wawancara
yang
dilakukan oleh M. Dinah Charlota Lerik
dan Yeni Damayanti pada 5 penduduk di
kota Kupang dan mendapati mitos yang
beredar
dikalangan
masyarakat
yaitu
berjemur matahari pada jam 10 pagi dapat
mencegah virus, mengkonsumsi temulawak
dapat mencegah virus dan orang berusia Universitas Nusa Cendana wawancara Universitas Nusa Cendana 299 Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Hubungan Antara Pengetahuan terhadap perilaku pencegahan COVID-19
pada mahasiswa papua dan papua barat di
Kupang
dengan
uji
staistik
Gamma
dengan batas
kemaknaan
dikatakan
bermakna apabila mempunyai p<0,05. Tabel 3. Karakteristik Responden
Berdasarkan Perguruan Tinggi
Perguruan Tinggi
Jumlah Persentase
(%)
Universitas Nusa Cendana Kupang
36
49
Politeknik Pertanian Negeri Kupang
7
9
Sekolah Tinggi Ilmu Kesehatan (STIKES)
Maranatha Kupang
3
4
Sekolah Tinggi Ilmu Kesehatan (STIKES)
Nusantara Kupang
2
3
Institut Agama Kristen Negeri (IAKN)
Kupang
25
34
Universitas Kristen Artha Wacana Kupang
(UNKRIS)
1
1
Total
74
100
Sumber : Data Primer Tabel 3. Karakteristik Responden
Berdasarkan Perguruan Tinggi Karakteristik Responden Tabel
1. Karakteristik
Responden
Berdasarkan Usia
Usia
Jumlah (n) Persentase (%)
17-25 Tahun
>25 Tahun
72
2
97
3
Total
74
100
Sumber : Data Primer Pada Tabel 1 dapat dilihat bahwa dari
74 responden yang diambil, berdasarkan
tabel di atas jumlah responden paling
banyak terdapat pada usia 17-25 tahun,
yaitu (97%). Dan jumlah responden yang
paling sedikit adalah usia >25 tahun, yaitu
(3%). Pada Tabel 3 dapat dilihat bahwa dari
74 responden yang diambil, berdasarkan
tabel di atas jumlah responden yang sedang
menempuh pendidikan di Universitas Nusa
Cendana sebanyak 36 orang (49%), jumlah
responden
yang
sedang
menempuh
pendidikan di Politeknik Pertanian Negeri
Kupang sebanyak 7 orang (9%), jumlah
responden
yang
sedang
menempuh
pendidikan di STIKES Maranatha sebanyak
3 orang (4%), jumlah responden yang
sedang menempuh pendidikan di STIKES
Nusantara sebanyak 2 orang (3%), jumlah
responden
yang
sedang
menempuh
pendidikan di IAKN sebanyak 25 orang
(34%) dan jumlah responden yang sedang
menempuh
pendidikan
di
UNKRIS
sebanyak 1 orang (1%). Tabel 2. Karakteristik Responden
Berdasarkan Jenis Kelamin
Jenis Kelamin
Jumlah(n)
Persentase (%)
Laki-laki
Perempuan
Total
36
38
74
49
51
100
Sumber : Data Primer Tabel 2. Karakteristik Responden
Berdasarkan Jenis Kelamin Tabel 2. Karakteristik Responden
Berdasarkan Jenis Kelamin Pada tabel 2. dapat dilihat bahwa dari
74 responden yang diambil, berdasarkan
tabel di atas jumlah responden dengan jenis
kelamin laki-laki sebanyak 36 orang, yaitu
(49%). Dan jumlah responden dengan jenis
kelamin perempuan sebanyak 38 orang,
yaitu (51%). Universitas Nusa Cendana 300 Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Hubungan Antara Pengetahuan Tabel 4. Hasil tabulasi silang hubungan antara pengetahuan terhadap perilaku pencegahan
COVID-19
Perilaku Pencegahan COVID-19
Total
Koefisien Korelasi
(r)
Nilai
p
Rendah
Sedang
Tinggi
Pengetahuan tentang
COVID-19
Rendah
0
0
0
0
0.587
*#0.078
Sedang
0
48
15
63
Tinggi
0
5
6
11
Total
0
53
21
74
Sumber : Data Primer Tabel 4. Hasil tabulasi silang hubungan antara pengetahuan terhadap perilaku pencegahan
COVID-19 Tabel 4. Hasil tabulasi silang hubungan antara pengetahuan terhadap perilaku pencegahan
COVID-19 didapatkan bahwa 11 mahasiswa memiliki
pengetahuan tentang COVID-19 dengan
kategori tinggi diantaranya lima mahasiswa
memiliki perilaku pencegahan COVID-19
dengan
kategori
sedang
dan
enam
mahasiswa memiliki perilaku pencegahan
COVID-19 dengan kategori tinggi. Pada tabel diatas didapatkan bahwa
dari
74
mahasiswa
tidak
terdapat
mahasiswa dengan pengetahuan tentang
COVID-19 dengan kategori rendah dan
perilaku pencegahan COVID-19 dengan
kategori rendah. Pada mahasiswa dengan
pengetahuan tentang COVID-19 dengan
kategori
sedang
dari
74
mahasiswa
didapatkan bahwa 63 mahasiswa memiliki
pengetahuan tentang COVID-19 dengan
kategori sedang diantaranya 48 mahasiswa
memiliki perilaku pencegahan COVID-19
dengan kategori sedang dan 15 mahasiswa
memiliki perilaku pencegahan COVID-19
dengan kategori tinggi. Pada mahasiswa
dengan pengetahuan tentang COVID-19
dengan kategori tinggi dari 74 mahasiswa Dari hasil uji statistik menggunakan
uji gamma, diperoleh hasil bahwa nilai p
0,078 (p<0,05) yang menunjukan bahwa
korelasi
antara
pengetahuan
tentang
COVID-19 terhadap perilaku pencegahan
COVID-19 tidak bermakna. Nilai korelasi
sebesar 0,587 menunjukan korelasi positif
dengan kekuatan korelasi yang sedang. 5. Hasil tabulasi silang hubungan antara sikap terhadap perilaku pencegahan
COVID-19. Perilaku Pencegahan COVID-19
Total
Koefisien Korelasi
(r)
Nilai p
Rendah
Sedang
Tinggi
Sikap tentang
COVID-19
Rendah
0
0
0
0
0.880
*#0.000
Sedang
0
47
7
54
Tinggi
0
6
14
20
Total
0
53
21
74
Sumber : Data Primer 19 dengan kategori sedang diantaranya 47
mahasiswa memiliki perilaku pencegahan
COVID-19 dengan kategori sedang dan 7
mahasiswa memiliki perilaku pencegahan
COVID-19 dengan kategori tinggi. Tabel 2. Karakteristik Responden
Berdasarkan Jenis Kelamin Pada
mahasiswa dengan sikap tentang COVID-
19
dengan
kategori
tinggi
dari
74
mahasiswa
didapatkan
bahwa
20
mahasiswa memiliki sikap tentang COVID- Pada tabel diatas didapatkan bahwa
dari
74
mahasiswa
tidak
terdapat
mahasiswa dengan sikap tentang COVID-
19 dengan kategori rendah dan perilaku
pencegahan COVID-19 dengan kategori
rendah. Pada mahasiswa dengan sikap
tentang COVID-19 dengan kategori sedang
dari 74 mahasiswa didapatkan bahwa 54
mahasiswa memiliki sikap tentang COVID- Universitas Nusa Cendana 301 Hubungan Antara Pengetahuan Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 gambaran pengetahuan mengenai deskripsi
umum
COVID-19,
sebagian
besar
responden (96%) sudah mengetahui bahwa
COVID-19 menyebabkan gejala gangguan
pernapasan akut seperti demam, batuk dan
sesak napas, sebagian besar responden
(92%) sudah mengetahui bahwa penularan
COVID-19 terjadi melalui kontak langsung
dan percikan yang keluar saat batuk dan
bersin, sebagian besar responden (76%)
yang percaya bahwa COVID-19 hanya
ditularkan oleh orang yang memiliki gejala,
sebagian kecil responden (24%) yang
mengira bahwa COVID-19 rentan pada
orang lebih muda, sedangkan sebagian
besar responden (66%) mengetahui bahwa
COVID-19 rentan pada orang yang lebih
tua dan orang-orang yang mempunyai
riwayat penyakit sebelumnya, separuh
responden (55%) memiliki pendapat bahwa
COVID-19 tidak berisiko terjadi pada
orang yang merokok dan mengonsumsi
NAPZA, sebagian kecil responden (28%)
memiliki pendapat bahwa jika sudah
menggunakan masker tidak perlu menjaga
jarak dengan orang lain, sebagian kecil
responden (16%) memiliki pendapat bahwa
orang sehat tidak perlu memakai masker
saat
keluar
rumah,
sebagian
besar
responden (89%) mengetahui bahwa orang
yang merasa kurang sehat sebaiknya
melakukan isolasi mandiri selama 14 hari,
separuh
responden
(47%)
memiliki
pendapat bahwa isolasi mandiri pada orang
yang baru pulang berpergian dari luar kota
tidak perlu dilakukan bagi yng tidak
memiliki gejala. 19 dengan kategori tinggi diantaranya enam
mahasiswa memiliki perilaku pencegahan
COVID-19 dengan kategori sedang dan 14
mahasiswa memiliki perilaku pencegahan
COVID-19 dengan kategori tinggi. Tabel 2. Karakteristik Responden
Berdasarkan Jenis Kelamin gambaran pengetahuan mengenai deskripsi
umum
COVID-19,
sebagian
besar
responden (96%) sudah mengetahui bahwa
COVID-19 menyebabkan gejala gangguan
pernapasan akut seperti demam, batuk dan
sesak napas, sebagian besar responden
(92%) sudah mengetahui bahwa penularan
COVID-19 terjadi melalui kontak langsung
dan percikan yang keluar saat batuk dan
bersin, sebagian besar responden (76%)
yang percaya bahwa COVID-19 hanya
ditularkan oleh orang yang memiliki gejala,
sebagian kecil responden (24%) yang
mengira bahwa COVID-19 rentan pada
orang lebih muda, sedangkan sebagian
besar responden (66%) mengetahui bahwa
COVID-19 rentan pada orang yang lebih
tua dan orang-orang yang mempunyai
riwayat penyakit sebelumnya, separuh
responden (55%) memiliki pendapat bahwa
COVID-19 tidak berisiko terjadi pada
orang yang merokok dan mengonsumsi
NAPZA, sebagian kecil responden (28%)
memiliki pendapat bahwa jika sudah
menggunakan masker tidak perlu menjaga
jarak dengan orang lain, sebagian kecil
responden (16%) memiliki pendapat bahwa
orang sehat tidak perlu memakai masker
saat
keluar
rumah,
sebagian
besar
responden (89%) mengetahui bahwa orang
yang merasa kurang sehat sebaiknya
melakukan isolasi mandiri selama 14 hari,
separuh
responden
(47%)
memiliki
pendapat bahwa isolasi mandiri pada orang
yang baru pulang berpergian dari luar kota
tidak perlu dilakukan bagi yng tidak
memiliki gejala. Dari hasil uji statistik menggunakan
uji gamma, diperoleh hasil bahwa nilai p
0,000 (p<0,05) yang menunjukan bahwa
korelasi
antara
pengetahuan
tentang
COVID-19 terhadap perilaku pencegahan
COVID-19
bermakna. Nilai
korelasi
sebesar 0,880 menunjukan korelasi positif
dengan kekuatan korelasi yang sangat kuat. Universitas Nusa Cendana PEMBAHASAN Berdasarkan hasil analisis didapatkan
bahwa pada mahasiswa papua dan papua
barat di kupang tidak terdapat pengetahuan
tentang COVID-19 dengan kategori rendah,
sikap tentang COVID-19 dengan kategori
rendah dan perilaku pencegahan COVID-
19 dengan kategori rendah. Berdasarkan
hasil analisis didapatkan bahwa pada
mahasiswa papua dan papua barat di
kupang didapatkan mahasiswa dengan
pengetahuan tentang COVID-19 dengan
kategori
sedang
memiliki
perilaku
pencegahan COVID-19 dengan kategori
sedang dan tinggi dan mahasiswa dengan
pengetahuan tentang COVID-19 dengan
kategori
tinggi
memiliki
perilaku
pencegahan COVID-19 dengan kategori
sedang dan tinggi. Berdasarkan hasil
analisis didapatkan bahwa pada mahasiswa
papua
dan
papua
barat
di
kupang
didapatkan
mahasiswa
dengan
sikap
tentang COVID-19 dengan kategori sedang
memiliki perilaku pencegahan COVID-19
dengan kategori sedang dan tinggi dan
mahasiswa dengan sikap tentang COVID-
19
dengan
kategori
tinggi
memiliki
perilaku pencegahan COVID-19 dengan
sedang dan tinggi. Dari keseluruhan gambaran sikap
tentang
COVID-19
mengenai
sikap
seseorang dalam menanggapi COVID-19,
hampir seluruh responden (97.3) setuju
bahwa mencuci tangan pakai sabun atau
menggunakan
handsanitizer
saat
memegang benda-benda di tempat umum,
hampir seluruh responden (96.8%) setuju
memakai masker saat berada di tempat
umum, sebagian besar responden (90.5%)
setuju menjaga jarak 1-2 meter dengan
orang lain saat berada di tempat umum,
sebagian besar responden (87.8) setuju Berdasarkan hasil analisis univariat
terhadap penilaian pengetahuan mahasiswa
menunjukkan bahwa rata-rata responden
menjawab pertanyaan dengan jawaban
yang
bervariasi. Dari
keseluruhan 302 Universitas Nusa Cendana Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 Hubungan Antara Pengetahuan untuk
menutup
mulut
dan
hidung
menggunakan lenga bagian atas bagian
dalam atau tisu saat batuk dan bersin,
hamper seluruh responden (93.2%) setuju
untuk mengisolasi diri selama 14 hari pada
saat baru pulang dari luar kota, sebagian
besar responden (77.1%) tidak setuju
memakai masker hanya digunakan pada
orang yag sakit sedangkan orang yang sehat
tidak perlu menggunakan masker, separuh
responden (55.4%) tidak setuju perokok
aktif dan pasif tiidak berisiko terkena
COVID-19,
sebagian besar
responden
(78.4%) tidak setuju untuk menerima tamu
atau berkunjung ke rumah orang tanpa
menggunakan
masker,
sebagian
besar
responden (73.0%) tidak setuju untuk
mengunjungi orang yang berusia 60 tahun
tanpa menggunakan masker, sebagian besar
responden (78.4%) tidak setuju untuk
berpergian ke tempatyang ramai tanpa
menggunakan masker. responden (78.4%) tidak menggunakan
fasilitas umum atau pergi ke tempat umum
(pasar, mall, tempat wisata dll) tanpa
menggunakan masker. Berdasarkan hasil analisis yang telah
dipaparkan, masih terdapat variasi pada
tingkat pengetahuan mahasiswa papua dan
papua barat di kupang. PEMBAHASAN Sikap tentang
COVID-19 pada mahasiswa papua dan
papua barat di kupang tergolong baik. Sedangkan perilaku pencegahan COVID-
19 pada mahasiswa papua dan papua barat
di kupang tergolong baik. Berdasarkan
hasil
uji
statistik
hubungan
antara
pengetahuan
tentang
COVID-19 terhadap perilku pencegahan
COVID-19 menggunakan uji Gamma,
didapatkan hasil uji berupa nilai p 0,078
untuk analisis bivariat menyatakan bahwa
pada penelitian ini tidak terdapat hubungan
yang bermakna antara pengetahuan tentang
COVID-19 terhadap perilaku pencegahan
COVID-19. Dari keseluruhan gambaran perilaku
pencegahan COVID-19 diketahui oleh
hampir seluruh responden yaitu dengan
mencuci
tangan
pakai
sabun
atau
menggunakan
handsanitizer
setelah
memegang benda-benda di tempat umum
(97.3%), hampir seluruh responden mandi
dan mengganti pakaian setelah pulang dari
berpergian
(97.3%),
hampir
seluruh
responden (97.3%) memakai masker saat
berada di tempat umum, hampir seluruh
responden (93.2) menutup mulut dan
hidung dengan lengan atas bagian dalam
atau tissue saat batuk dan bersin, hampir
seluruh responden (97.3%) menjaga jarak
1-2 meter dengan orang lain saat berada di
tempat umum, sebagian besar reponden
(62,2%) tidak berjabat tangan/ bersalaman
saat ertemu denga orang di tempat umum,
sebagian besar responden (68.9%) tidak
menghadiri acara yang mengumpulkan
banyak orang, sebagian besar responden
(82.4%)
tidak
menerima
tamu
atau
berkunjung
ke
rumah
orang
tanpa
menggunakan
masker,
sebagian
besar
responden (74.3%) tidak mengunjungi
orang tua yang berusia diatas 60 tahun
tanpa menggunakan masker, sebagian besar Berdasarkan
hasil
uji
statistik
hubungan antara sikap tentang COVID-19
terhadap perilaku pencegahan COVID-19
menggunakan uji Gamma, didapatkan hasil
uji berupa nilai p 0,000 untuk analisis
bivariat menyatakan bahwa pada penelitian
ini terdapat hubungan yang bermakna
antara sikap tentang COVID-19 terhadap
perilaku pencegahan COVID-19. Penelitian
sebelumnya
yang
dilakukan oleh Sukesih, dkk (2020) tentang
pengetahuan
dan
sikap
mahasiswa
kesehatan tentang pencegahan COVID-19
di Indonesia yaitu pengetahuan dan sikap
mahasiswa kesehatan tentang pencegahan
COVID-19 di Indonesia tergolong baik. Pada
penelitian
ini,
pengetahuan
mahasiswa papua dan papua barat di
kupang
tentang
COVID-19
bervariasi
berada dikategori sedang dan tinggi, sikap
mahasiswa papua dan papua barat di
kupang tentang COVID-19 tergolong baik
berada dikategori sedang dan tinggi dan
perilaku
pencegahan
COVID-19
pada 303 Universitas Nusa Cendana Hubungan Antara Pengetahuan Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 DAFTAR PUSTAKA mahasiswa papua dan papua barat di
kupang tergolong baik berada dikategori
sedang dan tinggi. mahasiswa papua dan papua barat di
kupang tergolong baik berada dikategori
sedang dan tinggi. 1. Direktorat Jenderal Pencegahan dan
Pengendalian
Penyakit
(P2P)
Kementerian Kesehatan RI. Pedoman
Pencegahan
dan
Pengendalian Coronavirus Disease
(COVID-19) Revisi ke-5. [Internet]
2020 [cited 13 Juli 2020] Available
from:https://covid19.go.id/p/protokol/
pedoman-pencegahan-dan-
pengendalian- coronavirus-disease-
covid-19-revisi-ke-5 1. Direktorat Jenderal Pencegahan dan
Pengendalian
Penyakit
(P2P)
Kementerian Kesehatan RI. Pedoman
Pencegahan
dan
Pengendalian Coronavirus Disease
(COVID-19) Revisi ke-5. [Internet]
2020 [cited 13 Juli 2020] Available
from:https://covid19.go.id/p/protokol/
pedoman-pencegahan-dan-
pengendalian- coronavirus-disease-
covid-19-revisi-ke-5 KESIMPULAN 1. Tidak
terdapat
hubungan
yang
bermakna antara pengetahuan dengan
perilaku
pencegahan
COVID-19
dengan
nilai
p=0.078
(p=0,000<0,05). 2. Terdapat hubungan yang bermakna
antara
sikap
dengan
perilaku
pencegahan COVID-19 dengan nilai
p=0.000 (p=0,000<0,05). 2. Kementerian Kesehatan RI. Keputusa
n Menteri Kesehatan Nomor HK.01.0
7/
MENKES/247/2020
tentang
Pedoman
Pencegahan
dan
Pengendalian Coronavirus Disease
2019 (COVID-19). 2020 2. 3. Pengetahuan
mahasiswa
tentang
COVID-19
dari
74
mahasiswa
didapatkan bahwa (85.1%) memiliki
pengetahuan dengan kategori sedang
dan (14.9%) memiliki pengetahuan
dengan kategori tinggi. 3. World Health Organization. WHO
Coronavirus Disease (COVID-19)
[Internet]
2020 [cited 26 Oktober 2020] Availa
ble from:https://covid19.who.int/?gcli
d=Cj0KCQjw59n8BRD2ARIsAAmg
PmLZhL1SfzJ6MKFPuvKVSOveOV
fhZ-
jLIQ1KBzvXG0JH49ywcakJbC8aAr
GtEALw_wcB 3. 4. Sikap mahasiswa tentang COVID-19
dari 74 mahasiswa didapatkan bahwa
(73.0%)
memiliki
sikap
dengan
kategori sedang dan (27%) memiliki
sikap dengan kategori tinggi. 5. Perilaku pencegahan COVID-19 dari
74 mahasiswa didapatkan bahwa
(71.6%)
memiliki
perilaku
pencegahan dengan kategori sedang
dan
(28.4%)
memiliki
perilaku
pencegahan dengan kategori tinggi. 4. Kementerian Kesehatan RI. Situasi
Terkini Perkembangan Coronavirus
Disease (COVID-19). [Internet] 2020
[cited 26 Oktober 2020] Available
from:
https://covid19.kemkes.go.id/situasi i
nfeksi emerging/infocoronavirus/situa
si-terkini-perkembangan-coronavirus-
disease-covid-19-26-oktober 2020
/#.X5caYPkzbIU Universitas Nusa Cendana Universitas Nusa Cendana Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 SARAN 1. Bagi subyek penelitian, diharapkan
dapat
meningkatkan
pengetahuan
tentang COVID-19 dan sikap tentang
COVID-19
sehingga
dapat
memperbaiki perilaku pencegahan
COVID-19 5. Susilo A, Rumende CM, Pitoyo CW,
et all. Coronavirus Disease 2019:
Tinjauan Literatur Terkini. Jurnal
Penyakit Dalam Indonesia | Vol. 7,
No. 1 . Maret 2020 5. 2. Bagi peneliti selanjutnya, diharapkan
dapat mengembangkan penelitian ini
dengan
meningkatkan
jumlah
responden dan melakukan penilaian
dengan melakukan observasi. Universitas Nusa Cendana 304 6.
Lerik MDC, Damayanti Y. Mitos
Covid-19 di Kalangan Masyarakat
Kota Kupang: Survei Cross-Sectional
Online.
Journal
of
Health
and Hubungan Antara Pengetahuan
Cendana Medical Journal, Edisi 22, Nomor 2, November 2021
6.
Lerik MDC, Damayanti Y. Mitos
Covid-19 di Kalangan Masyarakat
Kota Kupang: Survei Cross-Sectional
Online.
Journal
of
Health
and
Behavioral Science. Vol.2, No.2,
June 2020, pp. 130-137. Hubungan Antara Pengetahuan Cendana Medical Journal, Edisi 22, Nomor 2, November 2021 6. Lerik MDC, Damayanti Y. Mitos
Covid-19 di Kalangan Masyarakat
Kota Kupang: Survei Cross-Sectional
Online. Journal
of
Health
and Behavioral Science. Vol.2, No.2,
June 2020, pp. 130-137. Behavioral Science. Vol.2, No.2,
June 2020, pp. 130-137. 6. Lerik MDC, Damayanti Y. Mitos
Covid-19 di Kalangan Masyarakat
Kota Kupang: Survei Cross-Sectional
Online. Journal
of
Health
and Universitas Nusa Cendana 305
|
https://openalex.org/W3127387987
|
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Characterization of the ultrafine and fine particles formed during laser cladding with the Inconel 718 metal powder by means of X-ray spectroscopic techniques
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Spectrochimica acta. Part B, Atomic spectroscopy
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Characterization of the ultrafine and fine particles formed during laser
cladding with the Inconel 718 metal powder by means of X-ray
spectroscopic techniques Szilvia Kugler a,b,*, Attila Nagy a, J´anos Os´an b, L´aszl´o P´eter a, Veronika Groma b,
Simone Pollastri c, Alad´ar Czitrovszky a a Wigner Research Centre for Physics, POB 49, H-1525 Budapest, Hungary
b Centre for Energy Research, POB 49, H-1525, Budapest, Hungary
c Elettra Sincrotrone Trieste, Strada Statale 14 - km 163,5, I-34149 Basovizza, Trieste, Italy a Wigner Research Centre for Physics, POB 49, H-1525 Budapest, Hungary
b Centre for Energy Research, POB 49, H-1525, Budapest, Hungary
c Elettra Sincrotrone Trieste, Strada Statale 14 - km 163,5, I-34149 Basovizza, Trieste, Italy A R T I C L E I N F O Additive manufacturing is a rapidly growing industrial technology. Still, there is a lack of knowledge regarding
the fine particle emission and new particle formation during the processes and their consequences on the per
formance of the operation and the operator’s health as well. Therefore, we studied the properties of the emitted
particles during the 3D printing process using the Inconel 718 (Ni-based) superalloy. The number and the mass
concentrations were measured with a Scanning Mobility Particle Counter and Sizer. Size-fractionated samples
were collected by a cascade impactor, and the elemental composition of the particles was determined by total-
reflection X-ray fluorescence analysis, Scanning Electron Microscopy, Energy Dispersive Spectroscopy, and
microscopic X-ray fluorescence analysis in the different size fractions. The oxidation states of the metals (Cr, Mn,
Fe, Ni) in the samples were determined with the X-ray absorption near-edge structure (XANES) method. Most of
the particles were found in the ultrafine region with a size below 100 nm, and the mass size distribution had the
maximum at 85 nm. In the original powder, Ni was dominating with appr. 52 wt%, and the proportion of Cr was
around 20 wt%, and Mn was below 1 wt%. In the released particles, the Ni content decreased to appr. 26 wt%,
the Cr content increased to appr. 47 wt% and Mn increased to around 10 wt% for particles with a size between
0.07 and 10 μm. According to the XANES results, Cr, Mn and Fe were found to be oxidized significantly, whereas
Ni remained in the metallic form in the total emitted aerosol containing mostly ultrafine particles. The enrich
ment and oxidation of metals were correlated with each other. Keywords:
Directed energy deposition
Inconel 718
Ultrafine particles
X-ray analysis
Elemental composition
Oxidation state workpiece, where the metal particles are deposited, thus adding a new
layer onto the previously deposited ones. Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 Contents lists available at ScienceDirect * Corresponding author at: Wigner Research Centre for Physics, POB 49, H-1525 Budapest, Hungary.
E-mail addresses: kugler.szilvia@wigner.hu, kugler.szilvia@ek-cer.hu (S. Kugler). Available online 2 February 2021
0584-8547/©
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* Corresponding author at: Wigner Research Centre for Physics, POB 49, H-1525 Budapest, Hungary.
E-mail addresses: kugler.szilvia@wigner.hu, kugler.szilvia@ek-cer.hu (S. Kugler).
https://doi.org/10.1016/j.sab.2021.106110
Received 10 November 2020; Received in revised form 27 January 2021; Accepted 29 January 2021 Available online 2 February 2021
0584-8547/©
2021
The
Authors.
Published
by
Elsevier
B.V.
This
is
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access
article
under
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(http://creativecommons.org/licenses/by-nc-nd/4.0/).
https://doi.org/10.1016/j.sab.2021.106110
Received 10 November 2020; Received in revised form 27 January 2021; Accepted 29 January 2021 1. Introduction While the beam diameter of the YLM fiber
laser can be as small as 50 μm in the focal plane, the spot size on the
surface of the sample is adjustable from 0.48 to 3 mm, with the help of a
lens of 200 mm focal length, by a motorized beam expander. A low-
power visible diode laser marks the position of the infrared laser. In
the experiments, the laser’s measured output power was 361 W, and the
spot diameter was 1.04 mm. The powder nozzle developed and manufactured by OR Laser (Die
burg, Germany), in collaboration with the Fraunhofer Institute (Munich,
Germany), is in a coaxial configuration with the laser beam, and it is
mounted in a fixed vertical position. The nozzle for introducing the
shielding gas is in the powder nozzle after the glass window that protects
the focusing lens from contamination. In the present study, we have characterized the emitted aerosol
particles with respect to their dominating diameter range, morphology,
elemental composition and oxidation state of selected metals (Cr, Mn, Fe
and Ni) aiming to describe their enrichment/depletion and oxidation
compared to the original feedstock powder. The number and volume
distribution of the newly formed aerosol particles were determined by
scanning mobility particle counter and sizer (SMPS). Simultaneously,
total and size-fractionated aerosol samples were collected by a filter
pack and a cascade impactor, respectively. Morphology and elemental
composition of the particles were investigated by scanning electron
microscopy (SEM) and energy dispersive spectroscopy (EDS). Collective
elemental analysis of the aerosol particles was performed by total-
reflection and microscopic X-ray fluorescence methods (TXRF, μXRF). The oxidation state of selected metals in the aerosol particles were
studied non-destructively using X-ray absorption near-edge structure
method (XANES). The importance of the present study is that AM ma
chines can generate hazardous particles which can raise occupational
health issues for the operators of the machines. Furthermore, the
nanoparticles can influence the efficiency of the laser during operation. We used an open-table additive manufacturing machine for the experi
ments based on the directed energy deposition method, and the Inconel
718 nickel-based superalloy as feedstock powder. A GTV PF2/1 LC powder feeder with a 3.5 × 0.3 mm2 disk groove
and argon as the carrier gas fed the system with metal powder. We set
the powder focal plane to the surface of the substrate with a working
distance of 7 mm. 1. Introduction Laser-based additive manufacturing (AM) is a set of new technolo
gies where special machines build new parts using high-power lasers to
melt metallic particles to form new objects layer by layer. The so-called
3D metal printing technology can be effectively used in the production
of different parts for the aerospace, healthcare, energy, automotive, and
other industries. There are many different AM techniques, like binder
jetting, powder bed fusion, or directed energy deposition [1]. All of them
have their advantages and disadvantages in various applications. The
directed energy deposition method can be used to create a protective
coating on a surface, to repair parts, or to build new objects, too [2]. In
this technique, the intense laser beam locally melts the surface of the Many studies have been published about the effects of the process
parameters on the macro- and microstructure of the created new objects,
on their mechanical properties, porosity, surface quality, residual stress,
etc. [1] [2–5]. However, a relatively small amount of information is
available on the particle emissions during 3D metal printing processes. Concerning the indoor application of desktop 3D printers using, for
example, polylactic acid (PLA), acrylonitrile butadiene styrene (ABS)
thermoplastic, or polycarbonate (PC) feedstocks, people are aware of
their ultrafine particle emission [6–8]. Nevertheless, laser-based AM
machines are likely to generate airborne, nanoscale metal particles that
may cause lung or cardiovascular diseases [9,10]. The health effects of S. Kugler et al. Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 metal exposure of the lung are known to be a major concern, for
example, in the case of hard metal workers [11] or welders [12,13]. New
particle formation and the chemical composition of the plume were
studied for welding processes (e.g., [14,15]). Furthermore, the fraction
of the inhaled aerosol particles, which deposits in the lung, steeply in
creases with decreasing size in the ultrafine region [16]. Besides the
health issues, the particle emission may influence the building process as
well by absorbing and scattering a considerable amount of energy from
the process [15,17–19], and may distort the results of the on-line optical
measurements that aim to monitor the processes [20–22]. Therefore, it
is of crucial importance to characterize the particle emission of AM
processes and to explore its effects on the process itself and the health of
the operators. laser can operate in pulsed mode, we used it only in continuous wave
mode in our experiments. 1. Introduction The nominal mass flow rate of the metal powder
delivered by the powder feeder was 5.3 g/min. The measured carrier gas
flow rate was 3.8 L/min, and the shielding argon flow rate was 10 L/min
(at atmospheric pressure and room temperature). The substrate material
was a rolled 304L stainless steel plate with 10 mm thickness and a 100 ×
100 mm2 surface. The substrate with the workpiece was moved along the pre-
programmed trajectory under the powder nozzle using a 3D motorized
stage with a scan rate of 10 mm/s. The device is controlled by a microcomputer, which monitors and
controls the laser functions, the movements of the motorized stage, and
the powder feeder. With the ORLAS SUITE application, one can design
simple geometric shapes or import 3D objects created with a CAD soft
ware. After setting the laser and the trajectory parameters, it generates
G-code defining 2D and 3D processing paths in three or four axes. The
software transfers the G-code to the microcomputer, and the system
starts building the new part. Fig. 1 shows the schematic diagram of the
LRS EVO-Diodeline 450 laser welding machine equipped with the
powder nozzle and the powder feeder. While many industrial grade
additive manufacturing machines (3D metal printers) arrive fully
enclosed to the customers, open table systems provide more flexibility to
researchers, university laboratories, small/medium sized enterprises or
system integrators. 2.1. The additive manufacturing machine We used the LRS EVO-Diodeline 450 based open table additive
manufacturing machine from the O.R. Lasertechnologie GmbH (Die
burg, Germany) [23]. Table 1 summarizes the main features of the
system. i 2.2. Feedstock powder and substrate Superalloys are the workhorse material in the aerospace, petro
chemical, and nuclear industries where creep, corrosion, and heat
resistance are needed. Inconel 718 is a nickel-based superalloy. It is a
high-rupturing-strength material which offers toughness, corrosion The applied additive manufacturing machine consists of a fiber laser
which emits infrared radiation at 1070 nm, a powder nozzle that de
livers the shielding gas and the metallic powder to the workpiece, a four-
axis motorized stage to move the piece along pre-programmed trajec
tories, a powder feeder, and the controller unit connected to a computer. The laser source is a single-mode randomly polarized diode-pumped
Ytterbium YLM fiber laser (IPG Photonics, Oxford, USA) with 450 W
maximum mean output power. The laser power at the surface of the
workpiece is controllable between 10 and 100%. Although the fiber Fig. 1. Schematic diagram of the LRS EVO-Diodeline 450 based open table
additive manufacturing machine. Table 1
Technical data of the LRS EVO-Diodeline 450 laser welding machine. Table 1
Technical data of the LRS EVO-Diodeline 450 laser welding machine. 2.3. Description of the experiments The experiments took place in a small closed room of 26 m3, where
no extraction device was in operation during the measurements. The
LRS EVO-Diodeline 450 based additive manufacturing machine was
used to build demo rectangles of 15 × 15 × 5 mm3 (width × length ×
height) on a 304L stainless steel substrate (for details, see Sections 2.1
and 2.2). The production time of one rectangle was approximately 16
min. The powder feeder fed the machine with the MetcoAdd 718G metal
powder. The released particles spread in the room, mainly by Brownian
diffusion, which was locally influenced by thermally generated flows
around the hot metal. There was no ventilation in operation except the
fans of the laser machine below the motorized table where the work
piece was built. SMPS+C system, which was manufactured by the Grimm Aero
soltechnik Ltd. (Ainring, Germany), uses a Vienna-type differential
mobility analyzer (DMA) [27] to classify particles according to their
mobility diameter, and a condensation particle counter (CPC) as a de
tector to count them in each size bin. The size range, which is covered by
this instrument, spans from 10 to 1094 nm. The aerosol sample flow rate
is 0.3 L/min, and the sheath air flow rate is 3 L/min. The CPC uses n-
butanol as a working fluid and can measure concentrations up to 1.5 ×
105 particles/cm3 in single count mode and 107 particles/cm3 in
photometric mode. The size resolution of the instrument is 64 channels
per decade in scanning mode. The size distribution is determined by
scanning the DMA voltage and counting the particles with the CPC in
each size bin. The length of one scan was 4 min, which means that faster
processes may distort the measured size distribution. An SMPS (for details, see Section 2.4) was used to measure the
number concentration of the released aerosol particles. The SMPS’s
sampling point was 15 cm from the wall of the room and 80 cm from the
aerosol source. The length of the sampling line was 90 cm, and the
residence time of the particles in the sampling tube was 5 s. The instrument’s native output data is the number size distribution
based on the electrical mobility diameter. Its software calculates the
surface, volume, and mass size distributions from the number concen
trations in each size bin. 2.3. Description of the experiments Some assumptions apply for the calculations
like the particles are homogenous and spherical, and the user provides
the average density value. During the building process, size-fractionated aerosol samples were
also collected on 20 × 20 mm2 Si wafers and adhesive carbon substrates
with a 9-stage May-type cascade impactor (for details, see Section 2.5)
for 10 and 40 min sampling time for further morphological and chemical
analysis. li Table 3 In our experiments, we used the MetcoAdd 718G alloy from Oerli
kon. This metal powder has a size range from 45 to 90 μm, a spherical
form, and produced by the gas atomization manufacturing process. y
g
g
Table 3 shows the composition of 304L stainless steel, which was
used as the substrate to build demo rectangles on it. It is important to
highlight the Ni, Cr and Mn content of the 304L. 2.5. Cascade impactor sampling Furthermore, we used Teflon membrane filters (47 mm diameter and
1 μm pore-size) to sample the total suspended particles to determine
their elemental composition and oxidation state of the selected metals. Two samples were taken with 40 and 90 min sampling time and with a
flow rate of 16.7 L/min. New particles form from the metal vapor above the high-temperature
melt pool with a size mainly below 100 nm during the building of new
objects with the AM machine. Size-fractioned aerosol sampling was
performed with a 9-stage in-house developed extension of the May-type
cascade impactor [28] to be able to study the chemical and morpho
logical composition of particles not only in the fine but also in the ul
trafine size fraction (Fig. 2). Cascade impactors separate the aerosol
particles based on their inertia by increasing the speed of the airflow by
means of jet nozzles. Particles that cannot follow the airstream are
collected on plates via impaction (see Fig. 2). The area of the nozzles
decreases stage by stage, hence allowing the collection of particles of
consecutively decreasing diameters. The May-impactor has aero
dynamic cut-off diameters of 17.9, 8.9, 4.5, 2.25, 1.13, 0.57, 0.29, 0.18,
and 0.07 μm, for stages 1 to 9, respectively, at a flow rate of 16.7 L/min. Samples were collected on 20 × 20 mm2 Si wafers at stages 3 to 9 and on
adhesive carbon substrates at all stages except stage 1. The deposition
pattern of aerosol particles is a thin stripe with a 50 mm length and
widths decreasing with increasing stage number from 0.1 mm (stage 9)
to 1.8 mm (stage 2). Because of the high concentrations measured in the
laboratory during the AM process, the sampling time was short, 10 min
for Si substrates, and 40 min for carbon substrates. It should be noted
that the cascade impactor separates particles based on the aerodynamic
diameter that can differ from the electrical mobility diameter based on
the shape and density of the particles [29]. 2.4. Scanning mobility particle counter and sizer While the diameter of the MetcoAdd 718G grains is between 45 and
90 μm, the 3D printing process forms particles dissimilar in size to the
original ones, falling mainly in the ultrafine size range (below 100 nm). We used a scanning mobility particle sizer (SMPS) to measure the size
distribution and the concentration of these particles. The Model 5416 Table 2
Nominal composition of Inconel 718 [59]. Inconel 718
Element
Minimum wt%
Maximum wt%
Nickel (Ni)
50
55
Chromium (Cr)
17
21
Iron (Fe)
balance
Niobium (Nb)
4.75
5.5
Molybdenum (Mo)
2.8
3.3
Titanium (Ti)
0.65
1.15
Cobalt (Co)
N/A
1
Aluminum (Al)
0.2
0.8
Manganese (Mn)
N/A
0.35
Silicon (Si)
N/A
0.35
Copper (Cu)
N/A
0.3
Carbon (C)
N/A
0.08
Phosphorus (P)
N/A
0.015
Sulfur (S)
N/A
0.015
Boron (B)
N/A
0.006 Table 2
Nominal composition of Inconel 718 [59]. Table 2
Nominal composition of Inconel 718 [59]. Table 1 Technical data of the LRS EVO-Diodeline 450 laser welding machine. Laser type
Diode-pumped Ytterbium (Yb) Fiber Laser
Wavelength
1070 nm
Max. mean power (CW)
450 W
Max. pulse energy
45 J
Pulse peak power
4.5 kW
Pulse duration
0.5–50 ms
Pulse frequency
0.1–100 Hz
Focus diameter
0.05–2.0 mm
Operating mode
continuous wave or pulsed
Shielding/carrier gas
argon Technical data of the LRS EVO-Diodeline 450 laser welding machine. Laser type
Diode-pumped Ytterbium (Yb) Fiber Laser
Wavelength
1070 nm
Max. mean power (CW)
450 W
Max. pulse energy
45 J
Pulse peak power
4.5 kW
Pulse duration
0.5–50 ms
Pulse frequency
0.1–100 Hz
Focus diameter
0.05–2.0 mm
Operating mode
continuous wave or pulsed
Shielding/carrier gas
argon Fig. 1. Schematic diagram of the LRS EVO-Diodeline 450 based open table
additive manufacturing machine. 2 Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 S. Kugler et al. Table 3
Composition of 304L stainless steel. 304L
Element
Minimum wt%
Maximum wt%
Nickel (Ni)
8
12
Chromium (Cr)
18
20
Aluminum (Al)
N/A
0.1
Carbon (C)
N/A
0.03
Manganese (Mn)
N/A
2
Silicon (Si)
N/A
0.75
Phosphorus (P)
N/A
0.045
Sulfur (S)
N/A
0.03 resistance at elevated temperatures, and possesses outstanding weld
ability [24,25]. It can be used in a wide temperature range from −204 to
704 ◦C [26]. Table 2 shows the composition of Inconel 718. 2.8. Microscopic X-ray fluorescence analysis Measurements of the original feedstock powder and the collected
aerosol particles were performed on an in-house developed laboratory
μXRF setup [33], allowing point analysis and recording of 2D elemental
maps. Since the X-ray source cannot be moved, scanning is performed
through the movement of the sample across a stationary X-ray micro
beam. A low-power rhodium-anode X-ray source (iMOXS, IfG, Berlin,
Germany) [34] coupled with a polycapillary minilens (IfG, Berlin, Ger
many) was used to form a 20 μm microbeam [35]. Between the X-ray
tube and the polycapillary, a 25-μm Rh beam filter was used in order to
enhance Rh K characteristic lines in the excitation spectrum. In order to
detect the emitted characteristic X-rays, a Peltier-cooled Si drift detector
(SDD) with an active area of 30 mm2 (KETEK, Munich, Germany) was
used. The measurement process is computer-controlled by an in-house
developed LabView-based software specifically designed for the sys
tem [33]. It controls the sample stage, the spectrum acquisition process,
and displays the optical image of the sample. 2D elemental maps were recorded using a 10 μm step-size, and 3 s
dwell time per pixel on individual grains of the original metallic feed
stock powder. X-ray spectra were collected in the center positions of
individual grains, for 600 s. Longer measurement time was necessary for
the deposited aerosol particles. The recorded X-ray spectra were eval
uated by the AXIL software [31]. The system was calibrated using
metallic foils in the range of titanium to molybdenum. 2.9. X-ray absorption near-edge structure Since aerosol particles are deposited as a stripe,
the usual internal standardization is not straightforward; thus, the
quantification of elemental content was performed using calibration
based on external standards (Merck IV, 23 elements). The elemental
concentrations were calculated for each size fraction considering the
total length of deposited stripes (50 mm for each impactor stage) and the
collected air volume. Detection limits of 100 pg/m3 at each impactor
stage were reached using the system for transition metals in ambient
aerosol particles [32], assuring a reliable determination of elemental
size distributions in the present study. Spectra were collected using Si (111) monochromator in both
beamlines. At the XRF beamline, the monochromator was calibrated
before the measurements and spectra from size-fractionated aerosol
particles deposited on Si wafers were measured in TXRF geometry, using
an XFlash 5030 SDD (Bruker, Berlin, Germany). At the XAFS beamline, the energy calibration was accomplished by
collecting simultaneously a reference metal foil placed in a second
experimental chamber after the sample and after the I1 ionization
chamber. In this case, the geometry for fluorescence mode measure
ments on filter samples was standard 45◦/45◦, using an AXAS-M SDD
(KETEK GmbH, Munich, Germany) detector. XANES spectra were
collected also in transmission mode (sample at 90◦with respect to the
beam) on pure Cr, Mn, Fe and Ni metallic foils, stainless steel foils (301
and 316L) and reference compounds (Cr2O3, Fe2O3 and NiO) in the form
of pressed pellets. Stainless steel foils contain 16–19 wt% Cr, 6.0–13 wt
% Ni and 65–75 wt% Fe. All spectra were collected at room temperature both in air (XAFS
beamline) and in high vacuum (XRF beamline) conditions, using a
variable energy step as a function of the energy: Large step (5 eV) in the
first 200 eV of the spectrum, smaller step (0.2 eV) in the near-edge re
gion and a k-constant step of 0.03 Å−1 (up to 1.8 eV) further above the
absorption edge. The time per step was 5 s for fluorescence and 2 s for
transmission mode measurements. 2.6. Total-reflection X-ray fluorescence analysis The elemental composition of size-fractionated aerosol samples
collected on Si substrates was determined by total-reflection X-ray
fluorescence analysis (TXRF). A compact laboratory TXRF system [30] 3 S. Kugler et al. Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 Fig. 2. Schematic diagram of the May-type cascade impactor. S. Kugler et al. processing. Secondary electron images were recorded for studying the
particle morphology. The EDS analysis proved to be reliable by using an
acceleration voltage of 20 kV. 2.9. X-ray absorption near-edge structure In order to determine the oxidation state of selected metals in the
total and size-fractionated (stages 9 and 8) aerosol samples, X-ray ab
sorption near-edge structure (XANES) spectra were collected at the
Elettra synchrotron radiation facility (Trieste, Italy). Measurements
were performed at the K absorption edges of Cr, Mn, Fe, and Ni, both at
the XAFS [36] and the XRF [37,38] beamlines. Fig. 2. Schematic diagram of the May-type cascade impactor. was applied for the present study. A 50-W microfocus Mo-anode X-ray
tube (Petrick, Bad Blankenburg, Germany) was operated at 50 kV and 1
mA, and Mo-Kα X-rays were selected for excitation using a Mo/Si
multilayer monochromator (AXO, Dresden, Germany). Si wafer squares
were analyzed with aerosol deposit stripes horizontally, perpendicular
to the beam direction. X-ray spectra were recorded using a 7 mm2 silicon
drift detector (KETEK, Munich, Germany) with a round Zr collimator
and an analog signal processing unit (PGT, Princeton, USA). Measure
ments were performed in air. Counting time was set to 1600 s for stage 9
and 3000 s for the remaining stages. The AXIL software [31] was used to
evaluate X-ray spectra. Since aerosol particles are deposited as a stripe,
the usual internal standardization is not straightforward; thus, the
quantification of elemental content was performed using calibration
based on external standards (Merck IV, 23 elements). The elemental
concentrations were calculated for each size fraction considering the
total length of deposited stripes (50 mm for each impactor stage) and the
collected air volume. Detection limits of 100 pg/m3 at each impactor
stage were reached using the system for transition metals in ambient
aerosol particles [32], assuring a reliable determination of elemental
size distributions in the present study. was applied for the present study. A 50-W microfocus Mo-anode X-ray
tube (Petrick, Bad Blankenburg, Germany) was operated at 50 kV and 1
mA, and Mo-Kα X-rays were selected for excitation using a Mo/Si
multilayer monochromator (AXO, Dresden, Germany). Si wafer squares
were analyzed with aerosol deposit stripes horizontally, perpendicular
to the beam direction. X-ray spectra were recorded using a 7 mm2 silicon
drift detector (KETEK, Munich, Germany) with a round Zr collimator
and an analog signal processing unit (PGT, Princeton, USA). Measure
ments were performed in air. Counting time was set to 1600 s for stage 9
and 3000 s for the remaining stages. The AXIL software [31] was used to
evaluate X-ray spectra. 3.2. Elemental mass concentrations in the aerosol released during laser
cladding with metal powder SEM (for details see Section 2.7) was applied to determine the
morphology and the size distribution of the original MetcoAdd 718G
powder. Fig. 4 shows a typical SEM image for particles weakly attached
to an adhesive carbon tape. The measured size values agreed well with
the specifications of the feedstock powder, and most of the grains had a
spherical, and some of them had an elongated form. As a result of the gas
atomization manufacturing process, one can observe smaller satellite
grains stuck to the larger spherules with the size of a couple of microns. The elemental composition of the original MetcoAdd 718G powder
was determined with SEM/EDS and also using the μXRF technique (for
details see Sections 2.7 and 2.8). Fig. 5 shows the results of the two
different methods. The three most dominating elements are Ni appr. 52
wt%, Cr 20 wt%, and the Fe 18 wt%. The detected composition agrees
well with the nominal elemental composition of the Inconel 718 su
peralloy (see Table 2). li SEM (for details see Section 2.7) was applied to determine the
morphology and the size distribution of the original MetcoAdd 718G
powder. Fig. 4 shows a typical SEM image for particles weakly attached
to an adhesive carbon tape. The measured size values agreed well with
the specifications of the feedstock powder, and most of the grains had a
spherical, and some of them had an elongated form. As a result of the gas
atomization manufacturing process, one can observe smaller satellite
grains stuck to the larger spherules with the size of a couple of microns. Fig. 4. Secondary electron image of the MetcoAdd 718G grains. The elemental composition of the original MetcoAdd 718G powder
was determined with SEM/EDS and also using the μXRF technique (for
details see Sections 2.7 and 2.8). Fig. 5 shows the results of the two
different methods. The three most dominating elements are Ni appr. 52
wt%, Cr 20 wt%, and the Fe 18 wt%. The detected composition agrees
well with the nominal elemental composition of the Inconel 718 su
peralloy (see Table 2). li technique. Fig. 6 shows the elemental composition of the sampled
particles. Fig. 6 presents a different elemental ratio compared to the feedstock
material. The newly formed particles contain 42% of Cr, 29% of Ni, and
18% of Fe, which is significantly different from the original material. 3. Results 3.1. Number and mass concentrations of the aerosol particles released
during laser cladding with metal powder The SMPS (for details see Section 2.4) was detecting the number size
distribution of the newly formed aerosol particles using 4 min sampling
time with downward scanning mode. The results are shown in Fig. 3,
together with the calculated volume size distribution assuming spherical
particles. Fig. 4. Secondary electron image of the MetcoAdd 718G grains. Fig. 4. Secondary electron image of the MetcoAdd 718G grains. Based on the measured data, the vast majority of the formed particles
are below 100 nm, and more than 70% of the corresponding volume or
mass falls below 100 nm. The peak of the number size distribution is at
53 nm, the total concentration is 9.3 × 105 particles/cm3, and the mode
of the volume size distribution is at 85 nm (Table 4). We have to note
here that the particle concentration above the melt pool was beyond the
limit of the SMPS (107 particles/cm3). For the representative sampling
above the melt pool, we placed the probe to a 15 mm distance from the
source of the particles. Considering the high concentrations, the size
distribution of the particles near the melt pool is definitely different
from the one measured at 80 cm distance, i.e. particles are smaller and
the number concentration is higher. Table 4 Table 4
Statistical data of the size distribution based on SMPS measurements. Mean
(nm)
Mode
(nm)
Median
(nm)
Geometric
standard
deviation
Total
Number
42.44
53.28
43.42
1.68
930,000 (1/
cm3)
Volume
89.74
85.05
81.92
1.86
110.30
(μm3/cm3) Table 4
Statistical data of the size distribution based on SMPS measurements. 2.7. Scanning electron microscope and Energy Dispersive Spectroscopy The size-fractionated samples on the impactor plates were analyzed
by a scanning electron microscope (SEM) to determine the morphology
and composition of the particles. We used a Tescan MIRA3 scanning
electron microscope for this study. We analyzed the elemental compo
sition of the particles with Energy Dispersive Spectroscopy (EDS) pro
duced by EDAX Inc. and used the APEX™ Software for EDS for data For each sample, multiple spectra have been collected and merged in
order to increase the signal to noise ratio. The oxidation state of metals
in the aerosol samples was determined using least-squares Linear
Combination Fitting (LCF) based on reference spectra collected for
model compounds of known oxidation state. Background removal, 4 Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110
technique. Fig. 6 shows the elemental composition of the sampled
particles. Table 4
Statistical data of the size distribution based on SMPS measurements. Mean
(nm)
Mode
(nm)
Median
(nm)
Geometric
standard
deviation
Total
Number
42.44
53.28
43.42
1.68
930,000 (1/
cm3)
Volume
89.74
85.05
81.92
1.86
110.30
(μm3/cm3)
Fig. 4. Secondary electron image of the MetcoAdd 718G grains. Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 S. Kugler et al. normalization of XANES spectra as well as LCF were performed using the
Athena software package [39]. 3.2. Elemental mass concentrations in the aerosol released during laser
cladding with metal powder i
The measured morphology of the newly formed aerosol is shown in
Fig. 7. In the figure one can observe primary spherical particles with a
size in the order of 10 nm, and aggregates formed by these small Two samples were collected on Teflon membrane filters for the
whole size range of the particles [40] and were analyzed with the μXRF Fig. 3. Number and volume size distributions during cladding with powder measured in the fine fraction (below 1 μm). Fig. 3. Number and volume size distributions during cladding with powder measured in the fine fraction (below 1 μm). Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110
Fig. 5. Elemental composition of the original MetcoAdd 718G powder determined by SEM/EDS and μXRF. The error bars are the calculated standard deviation
values of 3 measurements. S. Kugler et al. S. Kugler et al. Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 Spectrochimica Acta Part B: Atomic Spectro Fig. 5. Elemental composition of the original MetcoAdd 718G powder determined by SEM/EDS and μXRF. The error bars are the calculated standard deviation
values of 3 measurements. the original MetcoAdd 718G powder determined by SEM/EDS and μXRF. The error bars are the calculated standard devia Fig. 6. Elemental composition of the aerosol particles released during the intensive laser-matter interaction (samples collected on Teflon membrane filters). of the aerosol particles released during the intensive laser-matter interaction (samples collected on Teflon membrane filters g. 6. Elemental composition of the aerosol particles released during the intensive laser-matter interaction (samples collecte particles. These aggregates have similar morphology to soot [41] and
flame-generated zirconia [42] aggregates. There are also self-standing
particles on the impactor plate with a diameter much below the size
range to which the impactor stage was rated. These particles are likely to
be formed as the debris of the larger ones that disintegrated upon the
collision with the impactor plate. Hence, the impacting conditions and
the diameter of the particles later observed on the collection substrate
surface cannot be correlated. 1.6–2.5 g/cm3 supporting that the majority of particles of 85 nm mass
median mobility diameter (see Fig. 3 and Table 4) are collected on the
smallest size class. The total mass concentration was 71.7 μg/m3 in the
whole covered size range (between 0.07 and 10 μm). The Ni-to-Cr ratio
changed from approximately 2 (original metal powder) to around 0.67
(new aerosol particles). 3.3. Oxidation state of metals by XANES Cr, Mn, Fe and Ni K-edge XANES spectra collected for aerosol 3.2. Elemental mass concentrations in the aerosol released during laser
cladding with metal powder 95% of the Cr and 89% of the Ni was in particles
between 70 and 180 nm. The Mn content was below 1 wt% in the
original powder, we measured 10 wt% for the particles of diameter
between 70 and 180 nm in the released aerosol. 1.6–2.5 g/cm3 supporting that the majority of particles of 85 nm mass
median mobility diameter (see Fig. 3 and Table 4) are collected on the
smallest size class. The total mass concentration was 71.7 μg/m3 in the
whole covered size range (between 0.07 and 10 μm). The Ni-to-Cr ratio
changed from approximately 2 (original metal powder) to around 0.67
(new aerosol particles). 95% of the Cr and 89% of the Ni was in particles
between 70 and 180 nm. The Mn content was below 1 wt% in the
original powder, we measured 10 wt% for the particles of diameter
between 70 and 180 nm in the released aerosol. Table 5 shows the measured mass concentrations of the selected el
ements in the different size fractions in terms of aerodynamic diameter
using the total-reflection X-ray fluorescence method. Fig. 8 shows the mass concentration of the elements measured with
different detection techniques between 70 and 180 nm size range. Both
methods show quite similar results. The oxygen is detected only with
SEM/EDS since the TXRF method was not performed under vacuum
conditions. ll
The TXRF analysis shows that 92% of the particles were between 70
and 180 nm in the size range covered by the cascade impactor, and the
mass concentration on this stage was 66.0 μg/m3. Based on calculations
provided for soot aggregates [41], the effective density was found to be
0.19 (uncompacted soot) to 0.30 (compacted soot) times the nominal
bulk density. Considering the nominal density of Inconel 718 alloy as
8.2 g/cm3, the effective density of the aggregates can be estimated as 3.3. Oxidation state of metals by XANES Cr, Mn, Fe and Ni K-edge XANES spectra collected for aerosol 6 Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 S. Kugler et al. Fig. 7. Secondary electron image of the MetcoAdd 718G particles collected for
the size range from 70 to 180 nm Si wafers. particles and metals (stainless steel) are summarized in Fig. 9. In order to
compare the spectra collected around K-edges of different metals, the
energy scale is displayed as relative to the respective K-edges (i.e. 3.2. Elemental mass concentrations in the aerosol released during laser
cladding with metal powder 5989
eV for Cr, 6539 eV for Mn, 7112 eV for Fe and 8333 eV for Ni). The
oxidation state can be derived from the chemical shift of the absorption
edge towards positive energies for oxidized chemical forms. Peaks and
oscillations near the absorption edges can also be used as fingerprints for
specific chemical states, e.g. a specific sharp and narrow peak at 4.4 eV
relative energy is characteristic for Cr6+ compounds [43]. i In general, XANES spectra of total aerosol on filter (XAFS beamline),
70–180 nm and 180–300 nm fractions (XRF beamline) look similar for
all selected metals. Because of the structural difference between pure
metals and alloys, [44–46] oscillations with smaller amplitude are
observed in the XANES spectra of metals in alloys. For this reason, Cr, Fe
and Ni K-edge XANES spectra collected on stainless steel 301 (contain
ing around 16–19 wt% Cr and 6–9 wt% Ni) were used for comparison
and further evaluation. Since the edge features (at around 0 eV relative) are smaller with
respect to the metallic spectra for Cr, Mn and Fe, the presence of
oxidized metals is significant in the aerosol samples (see Fig. 9). For Mn,
only a small step is visible at the nominal absorption edge energy of Mn
metal (0 eV relative), indicating that Mn is highly oxidized. For Cr, the
presence of Cr6+ could be excluded due to the absence of characteristic
pre-edge peak. Quantitative information on the oxidation state of Cr, Mn, Fe and Ni
was obtained by LCF performed using the standard spectra for Cr, Fe and
Ni in stainless steel (301), Mn metal and reference compounds of higher
oxidation states like Cr3+and Cr6+, Mn2+ and Mn3+, Fe2+ and Fe3+ as
well as Ni2+. The fitting results are summarized in Table 6. Fig. 7. Secondary electron image of the MetcoAdd 718G particles collected for
the size range from 70 to 180 nm Si wafers. Table 5 i
Among the four studied metals, Cr, Mn and Fe were found to be
oxidized significantly, whereas Ni remained in the metallic form in the
total aerosol. The order of metals based on oxidation degree was found
as Mn > Cr > Fe > Ni (see Table 6). Size-fractionated samples in the
70–180 nm and 180–300 nm diameter range contained metallic Cr and
Fe at the 79% and 77% level, respectively, significantly higher than in
the total aerosol samples. Taking into account the SMPS results as well,
the total aerosol collected by the filter is dominated by ultrafine particles
representing more than 70% of the total mass (see Fig. 3). It means that
the ultrafine aerosol particles contain the highest fraction of oxidized
Mn, Cr and Fe, so oxidation occurs despite the presence of the shielding
gas. Elemental mass concentrations in the aerosol released during laser cladding with
the MetcoAdd 718G metal powder. Elemental mass concentrations in the aerosol released during laser cladding with
the MetcoAdd 718G metal powder. t e
etco dd 7 8G
eta po de . Mass concentration (μg/m3)
Element
70–180 nm
180–300 nm
0.3–10 μm
Total 0.07–10 μm
Ni
17.30
0.40
1.60
19.30
Cr
29.00
0.60
1.00
30.60
Fe
11.90
0.30
0.70
12.90
Mn
6.90
0.10
0.10
7.10
S
0.15
0.10
0.49
0.74
Cu
0.27
0.01
0.02
0.30
Zn
0.03
0.00
0.00
0.03
Other
0.44
0.00
0.24
0.68
Total
66.0
1.5
4.2
71.7 Fig. 8. Mass concentrations of the elements in size range from 70 to 180 nm measured with TXRF and SEM/EDS 7
Fig. 8. Mass concentrations of the elements in size range from 70 to 180 nm measured with TXRF and SEM/EDS. Fig. 8. Mass concentrations of the elements in size range from 70 to 180 nm measured with TXRF and SEM/EDS. Fig. 8. Mass concentrations of the elements in size range from 70 to 180 nm measured with TXRF and SEM/EDS. 7 Spectrochimica Acta Part B: Atomic Spectrosc
Fig. 9. Comparison of Cr, Mn, Fe and Ni normalized XANES spectra of total aerosol collected on filter and metals. S. Kugler et al. Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 Fig. 9. Comparison of Cr, Mn, Fe and Ni normalized XANES spectra of total aerosol collected on filter and metals. pressure than Ni at 1200 ◦C temperature. 4. Discussion During the intense laser–metal interaction in the studied additive
manufacturing process, the evaporated atoms of the metallic parts mix
with the ambient gas, and the subsequent expansion of this mixture
leads to its cooling and supersaturation. As a result, new particles form
by nucleation and subsequent growth of the condensed-phase clusters
becomes possible. Table 6 LCF results for Cr, Mn, Fe and Ni K-edge XANES of aerosol particles, using
reference spectra of known oxidation states. LCF results for Cr, Mn, Fe and Ni K-edge XANES of aerosol particles, using
reference spectra of known oxidation states. LCF results for Cr, Mn, Fe and Ni K-edge XANES of aerosol particles, using
reference spectra of known oxidation states. Sample
Total aerosol on filter
(mostly ultrafine
particles)
70–180 nm
fraction
180–300 nm
fraction
Cr0 fraction
0.67 ± 0.01
0.79 ± 0.01
0.79 ± 0.05
Cr3+ fraction
0.33 ± 0.02
0.21 ± 0.03
0.21 ± 0.03
Cr6+ fraction
–
–
–
Cr mean
oxidation
number
þ0.97 ± 0.05
þ0.63 ±
0.10
þ0.64 ± 0.10
Mn0 fraction
0.10 ± 0.02
n.a. n.a. Mn2+ fraction
0.44 ± 0.02
n.a. n.a. Mn3+ fraction
0.46 ± 0.02
n.a. n.a. Mn mean
oxidation
number
þ2.26 ± 0.10
n.a. n.a. Fe0 fraction
0.74 ± 0.01
0.77 ± 0.01
0.77 ± 0.01
Fe2+ fraction
0.04 ± 0.01
0.10 ± 0.01
0.12 ± 0.01
Fe3+ fraction
0.22 ± 0.01
0.13 ± 0.01
0.11 ± 0.01
Fe mean
oxidation
number
þ0.73 ± 0.02
þ0.59 ±
0.05
þ0.57 ± 0.05
Ni0 fraction
0.96 ± 0.01
0.93 ± 0.01
0.93 ± 0.01
Ni2+ fraction
0.04 ± 0.01
0.07 ± 0.01
0.07 ± 0.01
Ni mean
oxidation
number
þ0.08 ± 0.02
þ0.14 ±
0.02
þ0.14 ± 0.02 The trend of enrichment in the ultrafine aerosol fraction is the same
as that of oxidation, i.e. Mn > Cr > Fe > Ni (Fig. 10). There is a hidden Fig. 10. Comparison of enrichment factor, mean oxidation number, volatility
and oxidation heat of metals in ultrafine aerosol at 1200 ◦C. Table 5 As the intense laser beam melts
the workpiece and the metal particles, first the Mn, then Cr and at last Ni
evaporate, the newly formed ultrafine particles contain Mn and Cr in
higher concentrations accordingly than in the original material. The
high apparent enrichment ratio makes it necessary to look for a sec
ondary source of the Mn besides the feedstock powder. One should note
that the substrate material contains Fe, Cr and small amount of Mn, that
can also play a role in enrichment when the first layer is built during the
AM process. The origin of the Mn can be the feedstock or the substrate
304L as well. The Inconel 718 contains only 0.35% and the substrate a
maximum of 2% of Mn. i 4.2. Occupational health effects of the released aerosol particles The chemical composition of the released aerosol particles can be
characterized by significant amounts of Fe, Cr, Mn, and Ni, where Ni, Cr,
and Mn are toxic metals [47–49]. An important feature of the ultrafine
metal oxide particles is the Mn enrichment, which, together with their
large amount, increase their toxic potential as a function of decreasing
particle size. The measured value of the Mn concentration was between
26 and 50 μg/m3, which reaches the occupational health limit value for
the respirable fraction (50 μg/m3) of this element in the European
Community [50]. Some countries, for example Germany has even a
more severe limit value for this fraction, namely 20 μg/m3.This metal
can damage the central nervous system and cause neuropsychiatric
disturbances [47]. According to several studies [51,52] Ni in metal form
is not considered as potentially carcinogenic component. Since 96% of
Ni was found in metallic form, serious health effects can be excluded
from the Ni. Some European countries have regulations for the occu
pational exposure limit values for Ni, but there is no uniform legislation. For example, in some countries the current limit value for the inhalable
fraction is 30 μg/m3, which is exceeded by the Ni concentrations
measured in the released aerosol. The SMPS measurements show that the majority of the particles were
in the ultrafine region with a size below 100 nm, and the mass size
distribution of the released particles shows a pronounced peak at 85 nm. The Ni-to-Cr ratio changed from approximately 2 (original metal pow
der) to around 0.67 (aerosol particles). Although the amount of Mn was
around or below the detection limit in the original feedstock powder, a
significant amount was released during laser cladding. 97% of Mn, 95%
of Cr and 89% of Ni were in particles in 70 to180 nm size range. The elemental compositions of the sampled aerosol particles were
determined through energy dispersive spectroscopy, total-reflection and
μX-ray fluorescence techniques, and the results were in good agreement. l
XANES results revealed that Mn, which metal was the most enriched
in the ultrafine aerosol fraction, was the most oxidized in the smallest
particulate fraction. Cr was also oxidized significantly, but the presence
of Cr6+ could be excluded. The order of metals based on formal oxida
tion number was found as Mn > Cr > Fe > Ni, and this order is also
relevant for the volatility of these metals. Declaration of Competing Interest The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper. 4.1. Elemental composition and oxidation states of selected metals in the
emitted particles The possible explanation for the enrichment of metals in the aerosol
compared to the original feedstock powder is that Cr has one order of
magnitude higher, and Mn has five orders of magnitudes higher vapor Fig. 10. Comparison of enrichment factor, mean oxidation number, volatility
and oxidation heat of metals in ultrafine aerosol at 1200 ◦C. 8 S. Kugler et al. Spectrochimica Acta Part B: Atomic Spectroscopy 177 (2021) 106110 causality relationship between these trends. The more volatile a
component is, the more it can enrich in the re-precipitated fraction of the
aerosol. And at the same time, the larger is its atomization ratio due to
the volatility, which provides an effective reactivity with the sur
rounding gases. Oxidation is due to the effective atomization, which is
the consequence of the volatility. This is an indirect evidence for that the
small particles do not form by the simple removal of the excess material
from large particles but an evaporation–precipitation mechanism is
effective. process. In the case of laser-based powder bed fusion of pure Zn powder,
the low densification of the created parts was attributed to the fluctu
ation of the laser energy deposited on the powder bed due to the
attenuation of the laser beam by small particles inside the evaporation
fume [56–58]. 5. Conclusions We characterized aerosol particles formed by vapor phase nucleation
during the interaction of intense laser beam and metallic alloys in laser
cladding processes. The size distribution, number and elemental mass
concentrations, the size-fractionated elemental composition and oxida
tion state of the metallic parts of the released ultrafine aerosol particles
were measured when an additive manufacturing machine was building
demo objects from nickel-based metal alloy powder. 4.2. Occupational health effects of the released aerosol particles Fortunately, no Cr6+ was detected, which is the most toxic and
carcinogenic form of this metal. The total Cr concentration varied from
134 to 179 μg/m3. The occupational health limit value for this compo
nent is 2 mg/m3 for 8 h in the inhalable fraction [53]. Although the
measured concentration is one order of magnitude lower than the limit
value, one should consider that the vast majority of the released parti
cles was found to be in the respirable fraction for which the limit values
are generally stricter if available. The Ni concentrations exceeded
whereas the Cr and Mn concentrations were close to or below the
currently valid limit values; therefore it is highly recommended for the
operators of the machine to wear suitable protective equipment and to
use extraction device. There are new recommendations for the European
Community to lower or set occupational health limit values for poten
tially toxic metal compounds in the respirable aerosol fraction [54]. The newly formed nanoscale metallic particles are potentially toxic
and/or carcinogenic to humans, which indicates the necessity of
applying proper extraction devices and/or personal protection
equipment. The new metal and metal oxide particles formed during the process
can cause considerable extinction of a fiber laser beam during cladding. Therefore, the metal vapor condensation effect should be taken into
account and monitored during the processes. 4.3. Effects of the released ultrafine particles on the quality of the AM
process 4.3. Effects of the released ultrafine particles on the quality of the AM
process 4.3. Effects of the released ultrafine particles on the quality of the AM
process The power stability of the energy source is an important factor in
additive manufacturing processes. The main contributions to the
attenuation of the laser power that reaches the surface of the substrate in
laser-based energy deposition processes are attributed to the reflection
from the substrate surface and the reflection and absorption by the
powder stream [55]. However, the power stability is influenced by the
generated ultrafine particles as well. While, in our study, the size of the
new particles was mainly below 100 nm, their measured number con
centration was in the order of 106 particles/cm3 at 80 cm, and above 107
particles/cm3 at 15 mm distance from the particle formation. Since these
nanoscale metal particles can absorb or scatter laser radiation, it affects
the quality of the beam focusing and the temporal power stability of the
laser radiation that reaches the surface. In the case of high power fiber
laser welding it was shown that the amplitude of the fluctuations of the
beam attenuation value could be higher than 10% when the beam
propagates through the generated aerosol-cloud above the melt pool
[18]. This effect can negatively influence the quality of the building CRediT authorship contribution statement Szilvia Kugler: Conceptualization, Methodology, Investigation,
Writing - original draft. Attila Nagy: Investigation, Writing - review &
editing, Funding acquisition. J´anos Os´an: Conceptualization, Method
ology, Investigation, Writing - review & editing, Funding acquisition. L´aszl´o P´eter: Investigation, Writing - review & editing. Veronika
Groma: Investigation, Writing - review & editing. Simone Pollastri:
Investigation, Writing - review & editing. Alad´ar Czitrovszky: Writing -
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Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect
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Abstract Over the last few decades, remarkable infrastructure growths have been noticed in secu-
rity-related issues throughout the world. So, with increased demand for Security, Video-
based Surveillance has become an important area for the research. An Intelligent Video
Surveillance system basically censored the performance, happenings, or changing infor-
mation usually in terms of human beings, vehicles or any other objects from a distance by
means of some electronic equipment (usually digital camera). The scopes like prevention,
detection, and intervention which have led to the development of real and consistent video
surveillance systems are capable of intelligent video processing competencies. In broad
terms, advanced video-based surveillance could be described as an intelligent video pro-
cessing technique designed to assist security personnel’s by providing reliable real-time
alerts and to support efficient video analysis for forensic investigations. This chapter deals
with the various requirements for designing a robust and reliable video surveillance system. Also, it is discussed the different types of cameras required in different environmental con-
ditions such as indoor and outdoor surveillance. Different modeling schemes are required
for designing of efficient surveillance system under various illumination conditions. Keywords: surveillance system, AIVSS, digital camera, types of camera, background
model, illumination Advance Intelligent Video Surveillance System (AIVSS):
A Future Aspect Mritunjay Rai, Agha Asim Husain,
Tanmoy Maity and Ravindra Kumar Yadav
Mritunjay Rai, Agha Asim Husain, Tanmoy M
and Ravindra Kumar Yadav Mritunjay Rai, Agha Asim Husain,
Tanmoy Maity and Ravindra Kumar Yadav
Additional information is available at the end of the chapter
Mritunjay Rai, Agha Asim Husain, Tanmoy Maity
and Ravindra Kumar Yadav
Additional information is available at the end of the chapter Additional information is available at the end of the chapter
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7,200 Provisional chapter © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
ttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
stribution, and reproduction in any medium, provided the original work is properly cited.
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
d
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d 1. Introduction In recent times, surveillance systems are gaining a lot of popularity. The government, various
organizations, residential societies, etc., are using these systems to keep a check on various activi-
ties for safety and security purposes. Earlier surveillance systems had a lot of dependence on © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. Intelligent Video Surveillance
2 2 human operators, it is lately that automated systems are being preferred because of their bet-
ter efficiencies and reliability [1]. It has been seen that surveillance with full human operators’
involvement has certain inadequacies like the high cost of labor, variations in long-duration cap-
turing and limited ability for multi-screen monitoring [2]. Traditional surveillance systems are
being complemented and even replaced by the advanced intelligent surveillance systems (AISS),
as the latter is used in identifying abnormal behavior and patterns in videos by developing arti-
ficial intelligence technologies, pattern recognition, and computer vision. This enables high accu-
racy monitoring of more scenarios by a few observers. In the last few years, the video surveillance
market has seen a major transformation into third generation video surveillance systems, mov-
ing to IP video from traditional analog video causing better processing power and improved
compression algorithm [3]. These Intelligent video surveillance systems are not just confined
to laboratories but have hit the marketplace as well. With this generation, the era of Intelligent
Video Surveillance began, not only in research labs but also in the marketplace. With the start
of 2010, many research labs, such as Kiwi Security Labs, started to broadcast the “Advanced
Intelligent Video Surveillance Systems” (AIVSS). With this production, a new category of fea-
tures is presented, which are expected to have a big impact on the marketplace security and a
sensor control. The Figure 1, shows an Intelligent Video Surveillance System. All the components
of the system are interconnected using many cameras for critical sites, by means of IP mega pixel
cameras. Selective ID protection feature has been provided in this architecture of AIVS. 1. Introduction Here, the disseminated keen design of the AIVS was used to execute the component Selective
ID Protection. Appropriately, the system could respect the current security law necessities in
a few nations, notwithstanding the prerequisites of governments and knowledge specialists
to ensure the character of their operators. Apart from the hardware (H/W) and software (S/W) which are considered as performance
improvisers and the architecture of inter-operational processing, the performance of the Figure 1. The distributed architecture of the advanced intelligent video surveillance system. Figure 1. The distributed architecture of the advanced intelligent video surveillance system. Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect 3 system depends upon the privacy of the system. Moreover, in many countries, privacy
issues are becoming more crucial and are considered as a performance decelerator. On one
side, the performance of the network depends upon the performance of each of Network
Element (NE), access performance, transmission performance, etc., which are also consid-
ered as performance decelerator and on the other side, the network’s performance depends
much upon the Security Management Process [4] of the advanced IVS system. Protection
assumes again a noteworthy part of security execution and in security administration forms
and accordingly on the system execution as an execution decelerator. From the perspective
of the Security Management Process, the suggestive development of process science was the
driving potential to build up an elite propelled IVS, which uses a keen Security Management
Process, which is controlling the system execution, i.e., system accessibility, secrecy and
trustworthiness, bringing about a substantial scale vital answer for security specialists and
governments [3, 4], Figure 2 demonstrates the execution effect of the progress smart video
surveillance system [5]. The remaining structure of the article is organized as the Section 2 deals with the basic require-
ments for designing of video surveillance system including different types of cameras and
video management systems using surveillance display. Section 3 discusses the surveillance
system for both indoor and outdoor environmental especially with illumination conditions. Section 4 discusses the different modeling schemes used for surveillance systems. Lastly,
Section 5 holds the conclusion and the future aspects. Figure 2. Performance impact on the distributed architecture of the advanced intelligent video surveillance system. Figure 2. Performance impact on the distributed architecture of the advanced intelligent video surveillance system. Intelligent Video Surveillance 4 2. Video surveillance system design requirements This section provides the details of decisions taken while designing the video surveillance
system. The design of a video surveillance system requires decisions that need familiarity
with the basic options and the basis behind the selection of any available choice in the mar-
ket [11]. So, designing a system requires better remote access, further remarkable mix with
different systems, enhanced picture quality and additionally that requires flexibility with
others system [12, 13]. In any case, for end clients to take the full preferred standpoint of
the advantages, the outline and execution of the arrangement should be precisely arranged
and executed. This will guarantee the system is adaptable and future-sealed and is proper
for a client’s need. These six stages cover guidance about choosing the correct hardware, an
assessment of the accessible innovation and help with the decisions that should be made. The following decisions are to be made for designing of video surveillance system are as
follows: 1. Camera and its type. 2. Video management system. 3. Types of video management system. 4. Storage type. 5. Types of video analytics. 6. Surveillance video display. 2.1. Camera and its type The camera position and
the type of cameras used under various conditions are important factors in video surveillance. These two parameters are briefly explained below: i. Positions for camera installation: Cameras should be placed in appropriate areas to
record relevant video. The appropriate areas for proper placement of cameras can be
entrances, hallways, driveways, T- Points, highway intersection points, exits, etc., and
in areas where there is a high density of people or vehicles. Moreover, cameras can be
placed in areas that require security such as parking spots, VIP areas, schools, restaurants
& hotels, bank locker rooms, hospitals, etc. Planting cameras at crucial and suitable points
is a cost-effective way to monitor and document people and vehicles arriving and depart-
ing certain facility. ii. Type of cameras to be used: There are many types of camera available on the market. The suitability of the camera depends upon the situation in hand. Fixed camera can be
used for recording only one specific view while a PTZ camera is generally used to cover
wider fields of views. Mostly fixed cameras are used in video surveillance as they are five
to eight times less costly than PTZ cameras. Color cameras are preferred during day time
and in highly illuminated areas. However, during night time and in poorly lit areas in-
frared or thermal cameras are used that gives black and white images. Thermal cameras
can also be used under settings of complete darkness, where they produce only contours
of objects. Cameras can be standard definition or high definition cameras that provide a
resolution of up to 16 MP. IP cameras digitize the recordings within the camera while analog cameras’ recordings (which
are used as surveillance cameras) are digitized on the computer. Video surveillance systems
usually make use of a combination of different type of cameras. Some of the camera types are
discussed briefly as under: a. PTZ camera: One of the commonly used camera for security purpose is PTZ camera;
where P stands for Pan, T for Tilt and Z for Zoom. Pan, Tilt, and Zoom are the main fea-
tures of this camera which is controlled by a software or via joystick. This security camera a. PTZ camera: One of the commonly used camera for security purpose is PTZ camera;
where P stands for Pan, T for Tilt and Z for Zoom. 2.1. Camera and its type In late 1990s, the digital cameras came into the market, they were built on Complementary
Metal Oxide Semiconductor (CMOS) based image sensor whose performance is better and are
cheaper than Charge-Coupled Devices (CCD). It has been seen during the last decade that on
an average there is an annual growth rate of around 12% of digital cameras throughout the
world. The credit can be attributed built-in intelligent image processing and pattern recogni-
tion algorithms. These smart digital cameras can spot motion, detect objects, read vehicle
number plates, and even identify human behaviors. They have become an essential compo-
nent to build active and automated control systems for many applications and will continue to
play a significant role in our daily life in the future [7]. Smart cameras are generally intended
to perform specific, repetitive, high-speed and high-accuracy jobs. The typical applications
of these smart cameras are Machine vision or intelligent video surveillance systems (IVSS). Video surveillance technology is functionally used in traffic cameras [9], which are used for
traffic footage recording and are many times shown during traffic reports on TV news. They
are placed over the traffic signals, along with the busy roads, and at busy junctures of the Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect 5 highway. Whether they record the movement of traffic for future study or to monitor traffic
and issue challans/tickets for any traffic rule violations, they are an extremely popular form
of video surveillance. They are commonly used in the monitoring & management of traf-
fic, computerized parking garages, driver support and control access systems, etc. License
Plate Recognition (LPR) is the most well-known and widely used application in the cate-
gory of traffic management and monitoring. Yet, due to increasing demand other categories
of vehicle classification have been added recently. Make and Model Recognition (MMR) &
Color Recognition (CR) of cars is a major and comparatively new functionality which helps
in detecting the model of the car, along with the vehicle types for, e.g., Light Motor Vehicle,
Heavy Motor Vehicle, etc. Installation of Camera plays an important role in the advance intel-
ligent video surveillance system. Cameras are the key contributors to the video surveillance system. The camera position and
the type of cameras used under various conditions are important factors in video surveillance. These two parameters are briefly explained below: Cameras are the key contributors to the video surveillance system. 2.1. Camera and its type Pan, Tilt, and Zoom are the main fea-
tures of this camera which is controlled by a software or via joystick. This security camera Intelligent Video Surveillance 6 has an ability to rotate 360 degrees so that it can cover a wide area and can zoom into
detail. The other features that attract toward this security camera are Weatherproof, Night
Vision, Multiple Alarms, Auto Focus, and Tamper Resistant. b. Box camera: Box Style security camera is an outdoor camera where customization of
the lens is possible. The lens can be variable or fixed. Box surveillance camera is an
ultra-high-resolution CCTV camera made with the new image sensor processor which
is capable of capturing video at 700 TV lines of resolution in color and black & white,
960H CCTV resolution. This box camera includes a 6-60 mm variable focal auto-iris lens
which gives security installers a lot of flexibility to adjust the camera angle of view and
zoom level. c. Dome camera: It is a combination of lens, camera and ceiling mount packaged in a dome
shape. This is well suited for surroundings that tend to get dirty, like kitchens and store-
rooms, etc., the best part of it is compact in size and artistically very attractive too. c. Dome camera: It is a combination of lens, camera and ceiling mount packaged in a dome
shape. This is well suited for surroundings that tend to get dirty, like kitchens and store-
rooms, etc., the best part of it is compact in size and artistically very attractive too. d. IP camera: An Internet Protocol camera generally transmits a digital signal using Internet
Protocol over a network. The main features of these security cameras are its high resolu-
tion and scalability. Right now, up to 30 Mega pixels are available in the market. d. IP camera: An Internet Protocol camera generally transmits a digital signal using Internet
Protocol over a network. The main features of these security cameras are its high resolu-
tion and scalability. Right now, up to 30 Mega pixels are available in the market. d. IP camera: An Internet Protocol camera generally transmits a digital signal using Internet
Protocol over a network. The main features of these security cameras are its high resolu-
tion and scalability. Right now, up to 30 Mega pixels are available in the market. e. 2.1. Camera and its type Wireless IP camera: As its name suggests that this type of camera is completely wireless,
installation is easy and reduces the rate of network cabling. The camera also has the fea-
ture of tilting and revolving which helps in maximum viewing with clarity and even in
low light conditions. Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect
7 f. Bullet camera: This security camera shaped like a bullet which is a combination of camera,
lens and packaged in a bullet style. This camera is good for dim light situations and can be
easily mounted to ceilings or walls because most of them use a tri-axis type of base. Bul-
let cameras come in all sizes (small, medium & large). Infrared bullet cameras generally
are larger in diameter to put up the extra space that their infrared Light Emitting Diodes
require. f. Bullet camera: This security camera shaped like a bullet which is a combination of camera,
lens and packaged in a bullet style. This camera is good for dim light situations and can be
easily mounted to ceilings or walls because most of them use a tri-axis type of base. Bul-
let cameras come in all sizes (small, medium & large). Infrared bullet cameras generally
are larger in diameter to put up the extra space that their infrared Light Emitting Diodes
require. f. Bullet camera: This security camera shaped like a bullet which is a combination of camera,
lens and packaged in a bullet style. This camera is good for dim light situations and can be
easily mounted to ceilings or walls because most of them use a tri-axis type of base. Bul-
let cameras come in all sizes (small, medium & large). Infrared bullet cameras generally
are larger in diameter to put up the extra space that their infrared Light Emitting Diodes
require. g. Day and night camera: This security camera is used for both indoor and outdoor envi-
ronments with low or dim illumination conditions. A day and night camera has distinc-
tive lenses that permit infrared emission formed by infrared LEDs and imitated from
objects to go through and reach to a Charge Coupled Device or Complementary MOS-
FET chip inside the camera. As a result, the end user can see the picture in total darkness
at the distance of infrared emission produced by LEDs. 2.1. Camera and its type A day and night camera can
have infrared LEDs mounted on its housing or can accept the emission, produced by
an infrared steeple. A Day and night cameras over and over again have changes in their
digital signal processor that pays for the alteration in illumination between day and
night methods. Intelligent Video Surveillance 8 h. Thermal camera (FLIR): The first commercial thermal imaging camera was used in 1965 for
high voltage power line inspections. Since then the utility of thermal imaging cameras for
industrial applications has become a pivotal market segment for FLIR (Forward-looking
IR) systems, a later name for high voltage power lines. The thermal imaging technology
has drastically evolved since then, and thermal imaging cameras have evolved to become
compact in size and look like a digital photo camera, they are now easy to use and produce
real-time crisp high-resolution images making them a widely important tool for industrial
applications [8]. They can detect anomalies that are generally invisible to the naked human
eye, thus taking corrective preventing costly systems going for a total breakdown. Thermal
imaging cameras are used to determine the maintenance requirements for electrical and
mechanical installations as they tend to generate unusual heat before they fail. Preventive
actions can be taken by discovering these hot-spots. A thermal imaging camera is a non-
invasive instrument which scans and visualizes the temperature distribution of surfaces
of a machine quickly and accurately, thus reducing cost and saving time across the world. 2.2. Video management system Video management system is the recording and management of access to the video, which is
captured by a camera and is then transferred to the module of the video surveillance system
[4]. There are two types of connections through which the captured video is transferred: i. Videos can be transmitted over the computer network IP or they can be sent as analog
videos. Videos from both IP cameras and analog cameras can be transferred over the
computer network whereas unlike analog cameras, IP cameras can connect directly to an
IP network. In case of analog cameras, an encoder must be installed to transmit analog
video over IP. The input from an analog camera is encoded and output a digital stream
for transmission over an IP network. ii. Depending upon whether IP camera or analog video camera is used, the captured video
can be transmitted over cables or through the air. Cables are generally considered inex-
pensive and the most reliable method of transferring video but, wireless is an important
alternative for transmitting videos as setting wires can be expensive for certain applica-
tions such as parking lots, fence lines, remote buildings, etc. 2.3. Types of video management system In a Video management system, videos taken by the cameras are stored, managed and are
transmitted to various viewers. The video management systems usually used in video sur-
veillance systems are: Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect 9 a. In a digital video recorder (DVR), videos are recorded from a surveillance camera on a
hard disk. It is a security system device in which the rate of the frame can be converted
from real-time to time lapse to save the disk space. They are more flexible as compared to
earlier analog VHS tape systems and allow easier transmission of video over a computer
network. Digital Video Recorders accepts only analog camera feeds as inputs and sup-
ports remote viewing over the Internet. DVR is a combination of software, hardware, and
video storage. b. Hybrid digital video recorders (HDVRs) support IP cameras. They can perform all the
functions of a digital video recorder mentioned above and adds support for IP and meg-
apixel cameras. c. Network video recorder (NVR) supports IP cameras only, however, to support analog
cameras it requires an encoder. NVR can record videos from a no. of digital CCTV cam-
eras that are transmitted over the network. c. Network video recorder (NVR) supports IP cameras only, however, to support analog
cameras it requires an encoder. NVR can record videos from a no. of digital CCTV cam-
eras that are transmitted over the network. d. IP video surveillance software is a product application that does not accompany any
equipment or capacity. The client must load and set up the PC/server for the prod-
uct which gives considerably more prominent opportunity and possibly bring down
cost yet in the meantime it accompanies noteworthy greater many-sided quality and
time important to set up and advance the system. IP video surveillance software is the
most regular decision for video systems that contain extensive camera tallies like at least
hundreds. 2.5. Video analytics type Video analytics encompasses the below-mentioned tasks: Video analytics encompasses the below-mentioned tasks: i. Storage optimization: Storage optimization is realized based on the motion detection. The video management systems decide to store the video when any motion/ moving ob-
ject [10] is spotted in the observed scene or else the video is either not stored or is stored at
a lower frame rate or a lower resolution to save storage space. Cameras may capture long
durations of inactivity when placed in buildings when they are locked, staircases, etc. This application helps in reducing the consumption of storage by 60–80% as compared to
continuous recording. ii. Identify threatening events: Video analytics can also be used to identify threatening
events to pro-actively identify any lapse in security incidents, be alert, and to stop them;
for example, license plate recognition, perimeter violation, abandoned objects detection,
and people counting. 2.4. Storage type In a video surveillance system, storage of the surveillance video is very vital. This video is
used for later retrieval and review. Cost of storage and security related fears specific to the
application of the video surveillance system determines the duration for which the video
should be stored [11]. For example, in supermarkets and restaurants video recordings are
kept for a relatively shorter duration as compared to the bank where there is a greater need to
hold videos for a longer duration (60–90 days) as there is a major threat of fraudulent investi-
gations that are often reported after many days of the incident. The digital data is stored per-
manently in the Storage, till it is purposely deleted. Even without power, this source holds its
content. Storage generally means magnetic disks, solid-state disks, and USB drives and may
also refer to magnetic tapes and optical discs like CDs, DVDs, etc. Although storage prices are
falling, the demand for the surveillance system and for the amount of storage is rising. Several
techniques have been developed to optimize the use of storage because of its high cost. There
are three main types of storage: i. Hard drives that are built inside a digital video recorder, network video recorder or serv-
er represents the internal storage. It is the most reasonably priced but may be less reliable
and scalable. Most frequently it is used in video surveillance systems and can provide a
storage of 2 TB to 4 TB. ii. Directly attached storage are the hard drives that are located outside of the digital video
recorder, network video recorder or server. It is more expensive as compared to internal
storage but has greater scalability, flexibility, and redundancy. Intelligent Video Surveillance 10 iii. Capacity clusters are IP based capacity places had some expertise in putting away video
gushing from an expansive number of cameras. They give proficient, adaptable and ver-
satile capacity. 3. Surveillance system and its types The word Surveillance has been derived from the French word “sur” means “from above” and
“veiller” means “to watch.” Surveillance means to monitor behaviors, movements, activities,
and information for controlling, managing, and protecting people. It can include observing
from assistance through an electronic device like CCTV cameras (Closed-circuit television)
or by keeping a track on electronically transmitted information like on phone calls & internet
traffic. It may also include a number of or relatively lesser technology means such as intelli-
gence agents, detectives, etc. Surveillance systems are readily being used by governments for
crime prevention and investigation, in intelligence gathering, and to protect people, objects,
processes, etc. For many, surveillance may be a violation of one’s privacy and has often been
criticized by many civil liberty activists. Laws in many countries have restricted their domes-
tic government & the private use of surveillance, generally restraining it to situations where
public safety is in jeopardy. Dictator government at times have any residential confinements, and universal secret activities
are normal among a wide range of nations. While observation systems have been exception-
ally ordinary for business properties, it took a while for them to wind up plainly mainstream
for private homes too. One reason is that wrongdoing insights demonstrate that the further
developed and cutting edge a security and observation system is, the more culprits will main-
tain a strategic distance from them through and through. Also, the cost of camera gear for
home utilization has altogether dropped as of late. The best sorts of observation system have
certain traits that you should give careful consideration to remember the ultimate objective to
ensure that you totally secure your property. One fundamental thing is that the system must
be effortlessly expandable to guarantee that as and when required you can cover more indoor
and outdoor regions with cameras. More established systems can be extremely restricted once introduced and will likewise just
permit a specific constrained measure of identification gadgets, including cameras, vibration,
and movement locators. This can turn out to be expensive if a system must be supplanted
because of extending business or private premises. Here are two or three signs on what to
pay uncommon identity to while exploring diverse sorts of security systems that will expand
insurance. 2.6. Display of surveillance video Videos captured by a surveillance system are eventually viewed by human beings and is usu-
ally used for past investigations. Some surveillance videos are watched online continuously,
e.g., in educational institutions to keep a check on student actions, in shops to keep an eye
on shoplifters and in public areas to identify criminal threats. Some surveillance videos are
viewed online infrequently by the owner of the apartment. Videos can be viewed in 4 differ-
ent ways: i. Local: It is viewed directly from the digital video recorder. Small facilities like Banks,
retail outlets, and small businesses ideally use the network video recorder to monitor
their sites. i. Local: It is viewed directly from the digital video recorder. Small facilities like Banks,
retail outlets, and small businesses ideally use the network video recorder to monitor
their sites. ii. Remote: It is viewed through standard remote PCs for viewing live and recorded videos
through an installed application, a web browser, or a powerful web viewing. iii. Mobile: This kind of viewing allows an instant check of the captured video. It holds great
importance in video surveillance systems. Mobile clients exist in the market for the last
few years, but there are challenges related to its implementation on PDAs/phones. How-
ever, a few latest technology phones have renewed interest in mobile viewing. iv. Video: Big security operation centers where a lot of cameras must be examined or scru-
tinized, video wall viewing is generally preferred. Video walls offer a very big screen so
that many people can watch the captured videos from a number of cameras at the same
time. They can change between numerous video streams and could automatically show
videos from points where alarms have been triggered. Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect
11 Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect
11 Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect
11 3.2. Indoor video surveillance There is no necessity for Indoor cameras to be weatherproof, that means they are easier to
install, are smaller with lesser restrictions. For indoor locations as shown in Table 1, a camera
that provides a very wide angle of view is needed so as reduce the requirement of the no of
cameras and to reduce the dark spots. Night vision and quality of the video captured are also
essential for Indoor video surveillance. 3.1. Outdoor video surveillance Outdoor video cameras play a very important role in law enforcement by not only capturing
video of potential criminals but also by preventing crimes. Past statistics on crimes show that
the more noticeable and better technology the camera systems are, the more it helps in pre-
venting criminal activities in business or residential buildings [6]. Factors that are necessary
for an outdoor camera are: they should be weather- resistant, and should include night vision
even in well-lit locations. This helps in ensuring that switching off the light may not affect the
camera captures. Cameras should be installed at a point capturing a wider angle & that is not
easily accessible from the ground. Intelligent Video Surveillance 12 3.3. Illumination and artificial lighting Amid no light conditions, it is not conceivable to see anything, yet to security cameras, they
are barely exceptional, some high fragile security cameras can get clear monochrome images Intruder detection
• Intrusion detection
• Object tracking
• Detection of an object in uncrowded scenes
Counting
• Statistical analysis
• Marketing
• Traffic flow analysis and reporting
• High accuracy
Nonmotion detection
• Detects static changes to a scene
• Handle crowded and busy environment
• Can detect tiny objects
• Can detect invisible object in low contrast
Crowd management
• Crowd management
• Traffic management
• Queue management
Table 1. Different application areas of video surveillance system. Table 1. Different application areas of video surveillance system. Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect 13 in starlight lighting up condition, they in like manner can see the object in whole darkness
while using additional infrared lighting. In this section, we offer the essential finding out
about the general lighting up, and the wide grouping of fake lighting. Lux is a prevented unit
in see from claiming lumen, and the lumen is a precluded unit in light from securing candela. The lumen (structure: lm) is the SI unit of luminous change, a measure of the vitality of light
obvious by the human eye and the candela is the SI base unit of luminous intensity. One lux
is equal to one lumen for each square meter, where 4n lumens is the total luminous change of
a light wellspring of one candela of luminous intensity.While picking a proper surveillance
camera to present, make a point to consider the illumination condition in the environment. Lux, it is the illumination level unit used to address the allocated domain illumination. In
incredible illumination region, the customer can use a general execution security camera. In
any case, the shading system for environmental illumination is under 2.0 Lux, a monochrome
illumination system for under 0.2 Lux environment use the higher execution security camera
(i.e., starlight security camera), which is extremely fundamental. Using Lux meter can evalu-
ate illumination level. If we do not have a lux meter, we can follow the general illumination
data table. Utilizing infrared LEDs to transmit vague (to human) infrared lights. The infrared light wave
length is 850 nm, which empowers camera to get monochrome images. 3.3. Illumination and artificial lighting While using the IR
illumination, the camera will encounter infrared-submersion issue because the photo setback
purposes of enthusiasm for objects arranged in central and short division watching an area. Remembering the true objective to handle this issue, IR sharp development was brought into
various security cameras. The Infrared splendid limit can modify camera’s Infrared inten-
sity as demonstrated by the watching objects, keep up a vital separation from IR-drenching
issue. Starting at as of late, the IR illumination can cover 0–200 meters independent. Using
white light LEDs to illuminate the area under observation. The white light LED wavelength is
450 nm, which has a place with noticeable light. The white light illumination can empower camera catch shading images in low illumination
or zero illumination environments. Compared with infrared illumination, white light illumi-
nation can work in particular application, for example, acknowledgment of vehicle number
plate. Moreover, the white light illumination can be utilized to stop interlopers/crooks. Sony
double light IP camera can naturally turn on white light illumination when individuals stroll
into observing territory. The white light has substantially shorter illumination separate than
infrared, its range is 0–50 m. Frequently, laser maker is set up into PTZ camera which offers
30× optical zoom limit. The bigger piece of laser maker utilizes 808 nm wavelength diodes,
laser maker has various purposes of intrigue; long detachment illumination, adaptability,
acclimate to the environment, long life expectancy. Laser illumination can help the camera
to get clear images with high clearness. Besides, it enables the camera to get pictures inside
1 kilometer or even 3 km long partitioned. In indoor surveillance, the cutting edge highlights liberal change revelation and following
estimation. Indoor conditions are passed on utilizing unmistakable truly little spaces that are
pulled back with dividers and give each other through passages and sections. In this condi-
tion, it is crucial to relate the district of a specific individual in various parts of the building
structure. It is less pivotal to track continually the difference in a man as this movement will Intelligent Video Surveillance 14 no vulnerabilities or conceivably buts break reliably because of building’s geology. In outside
surveillance, the cutting edge moreover merges veritable change request and following tal-
lies. The limit is that these figures are normally stunningly besides made than the relating
indoor checks on account of the distinctive quality presented by exceedingly factor lighting. 3.3. Illumination and artificial lighting The topology of outside conditions is correspondingly completely not exactly the same as
that of indoor conditions. Moving things are people and what more vehicles is in like manner,
going at all around higher speed. Snappier moving things require faster sorting out paces,
yet the figuring is liberally more computationally than those related to indoor surveillance
conditions. These repudiating necessities on a to a great degree basic level mean the specific
difficulties of a pushed outside security system. Utilizing Lux meter can check light level. If
you do not have a lux meter, you can propose Tables 2 and 3 to the running with general
illumination information. At the point when security camera works in entire darkness environment (i.e., 0 Lux), the
camera picture sensor would not have the capacity to catch images. In this condition, the
cameras have an artificial lighting system, for example, infrared LEDs, white light LEDs, and S. No. Places
Luminance intensity (in Lux)
1. Warehouse
20—75
2. Emergency passway
30—75
3. Corridor
75—200
4. Shop
75—300
5. Office
300—500
6. Bank
200—1000
7. Meeting room
300—1000
Table 2. Various indoor illumination conditions at different places. S. No. Places
Luminance intensity (in Lux)
1. Warehouse
20—75
2. Emergency passway
30—75
3. Corridor
75—200
4. Shop
75—300
5. Office
300—500
6. Bank
200—1000
7. Meeting room
300—1000
Table 2. Various indoor illumination conditions at different places. Table 2. Various indoor illumination conditions at different places. Table 2. Various indoor illumination conditions at different places. S. No. Places
Luminance intensity (in Lux)
1. Sunny
10,000—1,000,000
2. Cloudy
100—10,000
3. Dawn Twilight
1—10
4. Full moon over head
0.1—1
5. Quarter moon
0.01—0.1
6. Sunny Starlight
0.001—0.01
7. Cloudy Starlight
0.0001—0.001
Table 3. Various outdoor illumination conditions at different places.f
p Luminance intensity (in Lux) Table 3. Various outdoor illumination conditions at different places. 15 Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect also laser producers. These counterfeit lighting systems can help the camera to catch clear
monochrome or shading images in low illumination or zero illumination environments. As of
late, Hikvision and Sony propelled day and night security cameras which use double lighting
system. a. Background subtraction Foundation subtraction is generally utilized for recognizing moving articles from static cam-
eras. By evaluating the foundation, it would then be able to subtract it from the info outline,
by applying some limit esteem, to get the frontal area, i.e., the question. Diverse procedures
could be utilized to gauge the foundation, the easiest expect the foundation to be the past cas-
ing, and another probability is to apply a mean/middle channel for the keep going N outlines,
and accepting the foundation to be the outcome. This calculation is versatile to dynamic foun-
dation changes, simple to actualize and quick and pertinent for constant usage. Be that as it
may, the disadvantages are its reliance on the question speed, outline rate, colossal memory,
and above all, the edge utilized is neither worldwide nor time-invariant. A vigorous founda-
tion subtraction calculation ought to have the capacity to deal with lighting changes, monoto-
nous movements from the mess and long-haul scene changes [14, 15]. The accompanying
examinations influence utilization of the capacity of V(x,y,t) as a video to succession where t
is the time measurement, x and y are the pixel area factors. For example, V(1,2,3) is the pixel
power at (1,2) pixel area of the picture at t = 3 in the video grouping. A portion of the founda-
tion subtraction techniques are examined underneath: 4. Different strategies for smart video observation The present security system could be outlined as takes after: (a) security system act locally
and they do not participate in compelling way (b) high esteem resources are ensured defi-
ciently by obsolete innovation system. (c) Reliance on escalated human focus to identify and
survey dangers. Various strategies and calculations have been created and actualized, basi-
cally in programming, for question following, identification, and acknowledgment. A couple
of endeavors have been made to execute a portion of the calculations in equipment. Be that
as it may, those endeavors have not yielded ideal outcomes as far as exactness, power and
memory necessities. The decision of the ideal calculation can upgrade the execution and help
in settling these difficulties. Many question discovery calculations are by all accounts fantastic
applicants (e.g., difference-of-Gaussians (DoG), maximally stable extremal regions (MSER),
fully affine invariant feature detector (FIAF), scale invariant feature transform (SIFT), speeded
up robust features (SURF), background subtraction, and so on.), contrasting in their capacities
and prerequisites. 1. Using frame differencing A movement identification calculation starts with the division part where the frontal area or
moving items are sectioned from the foundation. The easiest method to execute this is to take Intelligent Video Surveillance 16 a picture as foundation and take the casings got at the time t, indicated by I (t) to contrast and
the foundation picture meant by B. Here utilizing basic number-crunching computations, we
can portion out the articles basically by utilizing picture subtraction method of PC vision
importance for every pixel in I(t), take the pixel esteem indicated by P[I(t)] and subtract it with
the comparing pixels at a similar position on the foundation picture meant as P[B]. In a mathematical equation, it is written as: P [F (t) ] = P [I (t) ] − P [B] The background is assumed to be the frame at time t. This difference image would only show
some intensity for the pixel locations which have changed in the two frames. Though we have
seemingly removed the background, this approach will only work for cases where all fore-
ground pixels are moving and all background pixels are static. A threshold “Threshold” is put
on this difference image to improve the subtraction (see Image thresholding). ∣P [F (t) ] − P [F (t + 1) ] ∣ > Threshold This implies the distinction picture’s pixels’ intensity is “thresholded” or sifted based on the
estimation of Threshold. The precision of this approach is reliant on speed of development in
the scene. Quicker developments may require higher edges This implies the distinction picture’s pixels’ intensity is “thresholded” or sifted based on the
estimation of Threshold. The precision of this approach is reliant on speed of development in
the scene. Quicker developments may require higher edges 2. Mean filter For figuring the picture containing just the foundation, a progression of going before pictures
arrive at the midpoint of. For figuring the foundation picture now t, B ( x, y, t ) = 1
__
N ∑
i=1
N
V(x, y, t − i ) where N is the quantity of going before pictures taken for averaging. This averaging alludes
to averaging comparing pixels in the given pictures. N would rely on the video speed (num-
ber of pictures every second in the video) and the measure of development in the video. In the
wake of figuring the foundation B(x, y, t) we would then be able to subtract it from the picture
V(x, y, t) at time t = t and limit it. In this manner the closer view is given as: where N is the quantity of going before pictures taken for averaging. This averaging alludes
to averaging comparing pixels in the given pictures. N would rely on the video speed (num-
ber of pictures every second in the video) and the measure of development in the video. In the
wake of figuring the foundation B(x, y, t) we would then be able to subtract it from the picture
V(x, y, t) at time t = t and limit it. In this manner the closer view is given as: ∣V (x, y, t) − B (x, y, t) ∣ > {Th} where Th is a threshold. Similarly, we can also use median instead of mean in the above cal-
culation of B(x, y, t). where Th is a threshold. Similarly, we can also use median instead of mean in the above cal-
culation of B(x, y, t). b. Speeded up robust features SURF is a scale-and pivot invariant intrigue point indicator and descriptor. The calculation
extricates striking focuses on the picture and registers descriptors of their surroundings that
are invariant to scale, turn and brightening changes. Nonetheless, identification and extrac-
tion are computationally requesting and consequently cannot be utilized as a part of systems
with restricted computational power [17]. a. Maximally stable extremal regions (MSER) The MSER calculation is an intrigue area identifier initially utilized as a part of wide-
standard stereo coordinating. MSER works on the information picture straightforwardly Advance Intelligent Video Surveillance System (AIVSS): A Future Aspect
17 with no smoothing, which brings about the location of both fine and coarse structures [16]. MSER performs all around contrasted with other nearby finders. The principle favorable
circumstances of the MSER recognition are that it is the speediest relative invariant area
locator. To the best of our insight, the main downside of the MSER is that its execution
debases with obscured pictures, which can be settled utilizing keen establishment of the
camera topology. 5. Discussion and conclusion The principal points of Advance Intelligent video surveillance system (AIVSS) are to build
up an observation system which can function as an indoor/open-air observation system. As
Advance Intelligent Video Surveillance System has a more extensive degree to take a shot at. As nowadays security and protection assume an essential part of the survival of the individ-
ual. The perfect observation engineering will have the accompanying attributes: elite, adapt-
ability, simple upgradability, low advancement cost, and a movement way to bring down
cost as the application develops and volume inclines. Also, the step by step expanding inno-
vations restricted the working of the Surveillance system, therefore the level of security must
be expanded with a specific end goal to stop the obstruction of interlopers. At present, the
video surveillance industry utilizes simple CCTV cameras and interfaces as the premise of
observation systems. These system parts are not effortlessly expandable and have low video
determination with practically zero flag preparing. Nonetheless, the up and coming age of
video surveillance systems will supplant these segments with more current computerized
LAN cameras, complex picture handling, and video-over-IP steering. They will never again
be essentially surveillance camera systems yet in addition video correspondence systems. The internet protocol (IP) based structure of the new surveillance systems takes into consider-
ation versatility, adaptability, and digital security. Different encoding and translating gauges
transport the video stream (MPEG4 CODEC is the standard utilized today). Other than the
CODEC work, picture pre-and post-handling improves the photo quality progressively with
low dormancy. Programmable rationale with inserted DSP squares, recollections, interfaces,
and off-the-rack IP arrangements enables a planner to meet the new system requirements. Security surveillance systems can be generally isolated into a few components, for example,
cameras, interchanges, stockpiling, picture preparing, and administration and back-end. Beginning with the camera, the present observation cameras are pushing toward the top-
quality period. Regardless of whether it is an IP camera that has turned out to be generally
acknowledged or HD-SDI cameras broadcasting in superior quality, they both can give up Intelligent Video Surveillance 18 to full 1080p HD determination surveillance pictures. Giving completely clear pictures is the
main role of shrewd surveillance, so top quality picture catch is fundamental, so the data gave
by the camera can be handled precisely. In this way, top notch cameras have turned into a key
part of observation system merchants. 5. Discussion and conclusion Be that as it may, cameras are only one a player in the
general surveillance system, and regardless of whether an ever-increasing number of cameras
and video encoders are incorporated, progressed and complex picture examination is still
performed on the backend, regularly using cloud-based preparing administrations. Author details Mritunjay Rai1*, Agha Asim Husain1, Tanmoy Maity1 and Ravindra Kumar Yadav2
*Address all correspondence to: er.mritunjayrai@gmail.com
1 Department of MME, IIT(ISM), Dhanbad, India
2 Department of ECE, SIET, Greater Noida, India Mritunjay Rai1*, Agha Asim Husain1, Tanmoy Maity1 and Ravindra Kumar Yadav2
*Address all correspondence to: er.mritunjayrai@gmail.com
1 Department of MME, IIT(ISM), Dhanbad, India
2 Department of ECE, SIET, Greater Noida, India 1 Department of MME, IIT(ISM), Dhanbad, India 2 Department of ECE, SIET, Greater Noida, India References [1] Aldasouqi I, Hassan M. Human face detection system using HSV. In: Proceedings
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Evaluating User Satisfaction of IT Services Through Service Quality Approach
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Corresponding Author Email: henoch.christanto@atmajaya.ac.id This research investigates user satisfaction
with Information Technology (IT) services at Unika Atma Jaya using the Service Quality
(Servqual) model, which includes Tangibles (X1), Reliability (X2), Responsiveness (X3),
Assurance (X4), and Empathy (X5) as variable. The analysis reveals that the Tangibles and
Reliability dimensions significantly contribute to positive user satisfaction, with respective
significant values of 0.000 and 0.001, along with corresponding T-table values of 4.197 and
3.323. This underscores the crucial role of tangible aspects, like facilities, and the reliability
of service delivery in enhancing overall user satisfaction. Conversely, the Responsiveness
dimension, with a significant value of 0.251 and a T-table value of 1.150, does not show a
statistically significant impact on user satisfaction, indicating that users' perceptions of
prompt service delivery may not be a decisive factor. Furthermore, the Assurance
dimension exhibits a significant negative impact, with a value of 0.000 and a T-table value
of -3.542, emphasizing the need for careful management in this area to prevent adverse
effects on overall user satisfaction. In conclusion, a focus on improving Tangibles and
Reliability dimensions, while addressing Assurance-related challenges, is vital for
optimizing the user satisfaction landscape at Unika Atma Jaya. Recommendations include
targeted enhancements in IT services in Tangibles and Reliability, specifically in
applications and on-campus facilities. These numerical findings serve as a strategic basis
for developing more effective, consistent, and responsive IT services to meet user
expectations in the future, particularly within the relevant unit (BSTI). Keywords:
service quality, Servqual, user satisfaction,
IT service Corresponding Author Email: henoch.christanto@atmajaya.ac.id Copyright: ©2024 The authors. This article is published by IIETA and is licensed under the CC BY 4.0 license
(http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.18280/isi.290225
ABSTRACT
Received: 18 September 2023
Revised: 15 January 2024
Accepted: 14 March 2024
Available online: 25 April 2024
Unika Atma Jaya, recognized as one of Indonesia's leading private universities, utilizes
information system technology to support both operational and academic activities. However, there is noticeable inconsistency in the implementation of this technology,
especially in effectively integrating it with relevant work units. Several existing information
systems fall short of meeting user satisfaction. This research investigates user satisfaction
with Information Technology (IT) services at Unika Atma Jaya using the Service Quality
(Servqual) model, which includes Tangibles (X1), Reliability (X2), Responsiveness (X3),
Assurance (X4), and Empathy (X5) as variable. The analysis reveals that the Tangibles and
Reliability dimensions significantly contribute to positive user satisfaction, with respective
significant values of 0.000 and 0.001, along with corresponding T-table values of 4.197 and
3.323. This underscores the crucial role of tangible aspects, like facilities, and the reliability
of service delivery in enhancing overall user satisfaction. Conversely, the Responsiveness
dimension, with a significant value of 0.251 and a T-table value of 1.150, does not show a
statistically significant impact on user satisfaction, indicating that users' perceptions of
prompt service delivery may not be a decisive factor. Furthermore, the Assurance
dimension exhibits a significant negative impact, with a value of 0.000 and a T-table value
of -3.542, emphasizing the need for careful management in this area to prevent adverse
effects on overall user satisfaction. In conclusion, a focus on improving Tangibles and
Reliability dimensions, while addressing Assurance-related challenges, is vital for
optimizing the user satisfaction landscape at Unika Atma Jaya. Recommendations include
targeted enhancements in IT services in Tangibles and Reliability, specifically in
applications and on-campus facilities. These numerical findings serve as a strategic basis
for developing more effective, consistent, and responsive IT services to meet user
expectations in the future, particularly within the relevant unit (BSTI). Keywords:
service quality, Servqual, user satisfaction,
IT service https://doi.org/10.18280/isi.290225
ABSTRACT Unika Atma Jaya, recognized as one of Indonesia's leading private universities, utilizes
information system technology to support both operational and academic activities. However, there is noticeable inconsistency in the implementation of this technology,
especially in effectively integrating it with relevant work units. Several existing information
systems fall short of meeting user satisfaction. Henoch Juli Christanto1*
, Lukas2
, Stephen Aprius Sutresno1
, Jenne Karolen1 1 Information System Department, Faculty of Engineering, Atma Jaya Catholic University of Indonesia, Jakarta 12930,
Indonesia 2 Cognitive Engineering Research Group, Faculty of Engineering, Atma Jaya Catholic University of Indonesia, Jakarta 12930,
Indonesia Ingénierie des Systèmes d’Information
Vol 29., No. 2, April, 2024, pp. 637-648 Journal homepage: http://iieta.org/journals/isi Evaluating User Satisfaction of IT Services Through Service Quality Approach Henoch Juli Christanto1*
, Lukas2
, Stephen Aprius Sutresno1
, Jenne Karolen1 1. INTRODUCTION platform (known as MyAtma) for course planning (Kartu
Rencana Studi, KRS), access to academic information through
the official website of Unika Atma Jaya, and Wi-Fi facilities
available at various locations on the Unika Atma Jaya campus. In the context of globalization and rapid advancements in
information technology, the use of Information Technology
(IT) in the educational environment has become increasingly
crucia [1, 2]. Higher education institutions, such as universities,
are increasingly reliant on IT systems and services to support
various academic and administrative activities [3]. At Atma
Jaya Catholic University (Unika Atma Jaya), a renowned
institution of higher learning, the implementation of quality IT
services is a key element in ensuring smooth operations and
user satisfaction [4]. However, this technology still shows
inconsistency, requiring further integration with related
functional units. Several information systems remain
underutilized, creating gaps in meeting user satisfaction [5]. The importance of user satisfaction assessment regarding IT
services is crucial in measuring the success of technology
implementation in the Unika Atma Jaya campus environment,
and it can also serve as feedback for BSTI to enhance IT
services further [6]. Therefore, this research aims to evaluate
the level of user satisfaction with IT services at Unika Atma
Jaya using a service quality approach method. This approach
details the evaluation based on generally recognized service
quality dimensions such as responsiveness, reliability,
assurance, empathy, and physical aspects (tangibles). Through the analysis of user satisfaction with IT services
using the service quality framework, it is expected to identify
detailed aspects that can be improved and points of excellence
that need to be maintained. Thus, the findings of this research
are anticipated to provide valuable input in efforts to enhance Biro Sistem Teknologi dan Informasi (BSTI) plays a key
role in overseeing various information systems, including the
Atma Jaya mobile application (known as AIDA) for
attendance tracking, the Oracle PeopleSoft Campus Solutions 637 expected variables [23]. The results of the respondent's
evaluation are processed to determine the Servqual score. The
Servqual model employs a multi-dimensional scale to assess
service
quality,
encompassing
customer
expectations,
perceptions, and gaps in order to measure and identify areas of
improvement [24, 25]. To obtain the desired data, the
operational research variables are shown in Table 1. the quality of IT services on this campus. Table 1. Research instrument variable Table 1. Research instrument variable 2.2 Academic information system Another study by Putri measured IT performance using the
Servqual method to explore the expected service perceptions
from the Information Systems Unit at University X. Despite
annual evaluations through balanced scorecards and
satisfaction questionnaires, the results were contradictory. Therefore, a reevaluation was conducted using the Servqual
method, concluding that immediate action was needed in
dimensions such as inadequate physical facilities like
hardware and software, and processes related to academic
activities, such as system access failures, card recording errors,
and student attendance list inaccuracies [27]. This research
contributes to the field by advocating for the use of the
Servqual method in measuring IT service performance at a
university to avoid incongruent results. The academic information system is designed to manage
educational information by applying hardware or software
technology so that the work process can be carried out
correctly and precisely and become helpful information. This
system's purpose is to support educational institutions'
management system so universities can provide more
appropriate, reasonable, and practical information services for
users [17]. 2.3 Service quality Service Quality (Servqual) is a service quality that can be
defined as the difference between the reality of the service
received by customers and customer expectations. [18]. The
Servqual method is a commonly used method for measuring
service quality [19]. The concept of service quality is an
evaluative factor that reflects consumer perceptions of five
specific aspects of service performance [20, 21]. The
satisfaction index with the Service Quality method is
Tangibles, Reliability, Responsiveness, Assurance, and
Empathy [22]. 2.4 Related works The previous research conducted by Maryana evaluated the
quality of the website of the Faculty of Economics and
Business at University X in Indonesia using the Importance
Performance Analysis (IPA) method. This study yielded the
highest performance score for the Information Quality variable
at 3.86 and the lowest for the Usability variable at 3.75 [26]. This research shares similarities with a study conducted in
terms of the research case, which is a university. However, this
study has a broader scope, focusing not only on the website
but also on various other applications and Wi-Fi services
within the IT service domain. Another difference lies in the
methodology employed, namely the Service Quality
(Servqual) method. The choice of the Servqual method for this
study was driven by its emphasis on customer satisfaction
measurement for each variable, whereas the IPA method
prioritizes variables based on importance and performance
values. 2.1 Information system satisfaction A system can be defined as a set of procedures, methods,
and ways of working that are carried out to achieve a specific
goal [10]. Information is a form of data processed to perform
a particular purpose. Data is still "raw" information and needs
to be processed further to be used and utilized [11, 12]. How
it is processed, and the type of data used depends on each
institution's description and needs [7, 13]. There are several
system characteristics, such as System components that make
up the system, also known as subsystems [14]. System
boundaries are parts that describe one system with another
plan [15]. This system limitation can also be called the system
boundary or scope [16]. 2. STATE OF THE ART Variable
Dimensions
Tangibles
Facilities, Equipment, Availability,
Format
Reliability
Fixed & Appropriate, Reliable, Thorough
& Accurate
Responsiveness
Handling complaints from system users,
Speed of information for users
Assurance
Proficiency, Trustworthiness, Integrity
Empathy
Ease of access, Comprehension
2.4 Related works An analysis is a study that uses information that can be used
to conclude, so it can be interpreted that analysis is an activity
of conducting a discussion on an object whose next stage is to
use data processing, after which conclusions can be drawn [7]. The analysis is also about studying existing systems to design
new plans or update existing ones [8]. From the above
definitions, we can conclude that analysis is the research stage
of studying a system, which aims to find problems that arise
and facilitate the system design stage—an activity to describe
and find solutions that someone does to look at an object in
detail [9]. 1. INTRODUCTION Furthermore, the
research findings are also expected to offer insights to other
higher education institutions facing similar challenges,
contribute to the continuous development of IT services in
higher education, and strengthen Unika Atma Jaya's position
as an innovative and responsive educational institution to
technological advancements. 3.1 Research methodology Research Stages to analyze IT service user satisfaction
using the Service Quality (Servqual) method, as set out in the
stages of the Figure 1. Servqual calculations are obtained by indicating each
variable. Either perception variables or expectation variables
were obtained from questionnaires distributed to consumers or The selection of SERVQUAL (Service Quality) as the
primary instrument for evaluating service quality and
satisfaction at Campus 3 of Atma Jaya University is based on 638 service quality and satisfaction at Atma Jaya University,
providing a solid foundation for a thorough understanding of
the delivery of IT services in the context of higher education. its excellence that distinguishes it from similar tools. SERVQUAL is known for its comprehensive and multi-
dimensional approach, covering five key dimensions that
detail critical aspects such as responsiveness, reliability,
assurance, empathy, and tangibles [18]. This excellence is
highly relevant when assessing the complexity of IT services
in a university environment. The customer-centric focus of
SERVQUAL provides a strong foundation, ensuring
assessments align with user expectations and perceptions,
particularly in the context of IT services in higher education. its excellence that distinguishes it from similar tools. SERVQUAL is known for its comprehensive and multi-
dimensional approach, covering five key dimensions that
detail critical aspects such as responsiveness, reliability,
assurance, empathy, and tangibles [18]. This excellence is
highly relevant when assessing the complexity of IT services
in a university environment. The customer-centric focus of
SERVQUAL provides a strong foundation, ensuring
assessments align with user expectations and perceptions,
particularly in the context of IT services in higher education. 3.2 Research hypothesis Based on the research scheme (see Figure 2), there are
variables
(X),
including
Tangibles,
Reliability,
Responsiveness, Assurance, & Empathy. And variable (Y)
consists of E-learning satisfaction (Y1), Atma Jaya Website
(Y2), MyAtma (Y3), AIDA Application (Y4), Computer (Y5),
and Wi-Fi (Y6). Another advantage that reinforces the selection of
SERVQUAL is its ability for benchmarking and gap analysis
[28]. This instrument allows for comparisons between
perceived and expected service quality, providing concrete
guidance for service improvements based on user needs [29]. The reliability and validity of SERVQUAL across various
industries affirm confidence in this instrument, while its
customizable and universal nature indicates its suitability for
evaluating IT services in a campus environment. Thus, the
choice of SERVQUAL has a positive impact on measuring 3.3 Questionnaire design The ability to access Wifi from different locations. Wifi can be accessed anytime. Reliable internet access, whether in the morning, afternoon, evening, or night. Table 4. Measurement of responsiveness
E- Learning
IT support quickly handles issues with the E-Learning website. Learning through the E-Learning website is easy and information is clear. Information about the content within the E-Learning website is quickly available. Website
IT support promptly addresses issues with the Atma Jaya website. Information about the University through the Atma Jaya website is easy to access and clea
Information about the content within the Atma Jaya website is quickly available. My Atma
IT support quickly handles issues with the MyAtma website. Learning through the MyAtma website is easy, and information is clear. Information about the content within the MyAtma website is quickly available. AIDA
IT support promptly addresses issues with the AIDA application. Learning through the AIDA application is easy, and information is clear. Information about the content within the AIDA application is quickly available. Computer
It's easy to contact IT support for computer-related issues. IT support quickly handles computer-related issues. The provided computers work smoothly. Wifi
IT support promptly addresses Wifi-related issues. It's easy to access Wifi. Th
id d Wifi
k
thl E-Learning
The E-Learning has an attractive color arrangement. The E-Learning website is easy for users to navigate due to its user-friendly layout. The E-Learning gives clear information through its features. Website
The website looks nice with its color scheme. The website is user-friendly with an easy-to-use layout. The features inside the website provide clear information. My Atma
The MyAtma website has an appealing color arrangement. The user interface of the MyAtma is designed to be user-friendly. The features inside the MyAtma provide clear information. AIDA
The AIDA app looks nice with its colors. The AIDA app is easy to use with a simple layout. The features in the AIDA app give clear information. Computer
Computers are available according to user needs. Computer placements are in strategic locations. Computer facilities work well. Facilities in the lab room provide comfort for users. Wifi
Wifi is available as per user needs. Wifi placements are in strategic locations. Wifi facilities work well. Table 3. Measurement of reliability
E- Learning
The website provides accurate information to users. Timeliness in delivering relevant information for users. The ability to access the E-Learning website from different locations. The E-Learning website can be accessed anytime. 3.3 Questionnaire design Surveys can comprise closed-ended or open-ended
questions or statements, which can be administered directly to
participants or distributed through various channels such as
social media or the internet [30]. The Measurement can be
seen from Tables 2-7. Figure 1. Stage of research methodology
Figure 2. Scheme of research
639 Figure 1. Stage of research methodology Figure 1. Stage of research methodology Figure 2. Scheme of research Figure 2. Scheme of research Figure 2. Scheme of research 639 Table 2. Measurement of tangible E-Learning
The E-Learning has an attractive color arrangement. The E-Learning website is easy for users to navigate due to its user-friendly layout. The E-Learning gives clear information through its features. Website
The website looks nice with its color scheme. The website is user-friendly with an easy-to-use layout. The features inside the website provide clear information. My Atma
The MyAtma website has an appealing color arrangement. The user interface of the MyAtma is designed to be user-friendly. The features inside the MyAtma provide clear information. AIDA
The AIDA app looks nice with its colors. The AIDA app is easy to use with a simple layout. The features in the AIDA app give clear information. Computer
Computers are available according to user needs. Computer placements are in strategic locations. Computer facilities work well. Facilities in the lab room provide comfort for users. Wifi
Wifi is available as per user needs. Wifi placements are in strategic locations. Wifi facilities work well. Table 3. Measurement of reliability
E- Learning
The website provides accurate information to users. Timeliness in delivering relevant information for users. The ability to access the E-Learning website from different locations. The E-Learning website can be accessed anytime. Website
The website provides correct information to users. Timely delivery of relevant information for students. The ability to access the Atma Jaya website from different locations. The Atma Jaya website can be accessed anytime. My Atma
The website provides accurate information to users. Timely delivery of relevant information for users. The ability to access the MyAtma website from different locations. The MyAtma website can be accessed anytime. AIDA
The application provides accurate information to users. Timely delivery of relevant information for users. The ability to access the AIDA application from different locations. The AIDA application can be accessed anytime. Computer
Users can easily connect to computers. Computers can be accessed anytime. Wifi
Users can easily connect to Wifi. 3.3 Questionnaire design My Atma
The MyAtma website aligns with the user's access rights. It's comfortable to use the MyAtma website. The MyAtma website meets user needs. Trust and confidence in attached documents. AIDA
The AIDA application aligns with the user's access rights. It's comfortable to use the AIDA application. The AIDA application meets user needs. Trust and confidence in attached documents. Computer
Computers align with the user's access rights. Comfortable use of the provided computers. Wifi
Wifi aligns with the user's access rights. Comfortable use of Wifi. Table 6. Measurement of empathy
E- Learning
The E-Learning website can be accessed by users of all ages. IT Support provides information/understanding to users about the E-Learning website facing issues. Website
The Atma Jaya website can be accessed by users of all ages. IT Support provides information/understanding to users about the Atma Jaya website facing issues. My Atma
The MyAtma website can be accessed by users of all ages. IT Support memberikan informasi/pemahaman kepada pengguna tentang situs Web MyAtma yang mengalami masalah
AIDA
The AIDA application can be accessed by people of all ages. IT Support provides information/understanding to users about the MyAtma website facing issues. Computer
The AIDA application can be accessed by users of all ages. IT Support provides information/understanding to users about computers facing issues. Wifi
Wifi can be accessed by users of all ages. IT Support provides information/understanding to users about Wifi facing issues. Table 7. Measurement of satisfaction
Satisfaction
The website/application is easy and clear for users to use. The user interface of the website/application is easy to understand and not confusing for users. The website/application can be accessed anytime by users. Users feel safe and comfortable when accessing IT services (Software & Hardware). Computer and Wifi facilities work well. Users can easily connect to computers and Wifi. IT Support responds when users experience issues with the use of IT services (Software & Hardware). Table 7. Measurement of satisfaction The measurement scale used to generate quantitative data is
the Likert Scale. The Likert Scale measures individual
agreement or disagreement with program plans, program
performance, or the level of program success [31]. The Likert
Scale is also used to measure individual or group perceptions,
attitudes, or opinions about an event (see Table 8). studied by researchers and conclusions drawn about the people
[32]. 3.3 Questionnaire design The population in this study are active users of Unika
Atma Jaya IT services, such as Lecturers and Active Students,
for the population of active users of Unika Atma Jaya IT
services is up to 10,000 people. The sample is called part of the total population in a study. The following are the sample criteria for this study: 1. Active
users of IT services at Unika Atma Jaya; 2. Have used IT
services in terms of software and hardware at Unika Atma Jaya;
3. Respondents are willing to become research subjects. Table 8. Question score according to likert scale Table 8. Question score according to likert scale
Number
Option
Score
1. Strongly Agree
5
2. Agree
4
3. Neutral / Uncertain
3
4. Disagree
2
5. Strongly Disagree
1
3.4 Data collection Number
Option
Score
1. Strongly Agree
5
2. Agree
4
3. Neutral / Uncertain
3
4. Disagree
2
5. Strongly Disagree
1
a collection This research employs a probability-based sample selection
method. Probability sampling holds a particular advantage in
research methodology due to its capability to afford each
element in the population a known and non-zero chance of
inclusion in the sample [33, 34]. This ensures equitable
representation, allowing researchers to generalize findings to
a broader population with a higher level of confidence [35]. The sample collection is based on feedback from
questionnaires distributed through university mail. The 3.3 Questionnaire design Website
The website provides correct information to users. Timely delivery of relevant information for students. The ability to access the Atma Jaya website from different locations. The Atma Jaya website can be accessed anytime. My Atma
The website provides accurate information to users. Timely delivery of relevant information for users. The ability to access the MyAtma website from different locations. The MyAtma website can be accessed anytime. AIDA
The application provides accurate information to users. Timely delivery of relevant information for users. The ability to access the AIDA application from different locations. The AIDA application can be accessed anytime. Computer
Users can easily connect to computers. Computers can be accessed anytime. Wifi
Users can easily connect to Wifi. The ability to access Wifi from different locations. Wifi can be accessed anytime. Reliable internet access, whether in the morning, afternoon, evening, or night. Table 4. Measurement of responsiveness
E- Learning
IT support quickly handles issues with the E-Learning website. Learning through the E-Learning website is easy and information is clear. Information about the content within the E-Learning website is quickly available. Website
IT support promptly addresses issues with the Atma Jaya website. Information about the University through the Atma Jaya website is easy to access and cle
Information about the content within the Atma Jaya website is quickly available. My Atma
IT support quickly handles issues with the MyAtma website. Learning through the MyAtma website is easy, and information is clear. Information about the content within the MyAtma website is quickly available. AIDA
IT support promptly addresses issues with the AIDA application. Learning through the AIDA application is easy, and information is clear. Information about the content within the AIDA application is quickly available. Computer
It's easy to contact IT support for computer-related issues. IT support quickly handles computer-related issues. The provided computers work smoothly. Wifi
IT support promptly addresses Wifi-related issues. It's easy to access Wifi. The provided Wifi works smoothly. 640 Table 5. Measurement of assurance E- Learning
The E-Learning website aligns with the user's access rights. It's comfortable to use the E-Learning website. The E-Learning website meets user needs. Trust and confidence in attached documents on the E-Learning website. Website
The Atma Jaya website aligns with the user's access rights. It's comfortable to use the Atma Jaya website. The Atma Jaya website meets user needs. Trust and confidence in attached documents. 𝑛=
1.962 × 0.5(1 −0.5) × 10,000
0.052(10,000 −1) × 1.962 × 0.5(1 −0.5)
𝑛= 369.98
(2) minimum required sample size is calculated using a cross-
sectional approach, as depicted in Eq. (1): minimum required sample size is calculated using a cross-
sectional approach, as depicted in Eq. (1): (2) 𝑛=
𝑍1−𝛼/2
2
𝑃(1 −𝑃)𝑁
𝑑2(𝑁−1)𝑍1−𝛼/2
2
𝑃(1 −𝑃)
(1) The result of the sample calculation using the cross-
sectional formula yielded a sample size of 369.98 respondents,
rounded up to 370 respondents. For the questionnaire
distribution, user information such as faculty, gender, and
region was collected. The total number of respondents in the
questionnaire distribution was found to be 372 respondents. (1) Keterangan: 𝑛: jumlah sampel 𝑍: skor z pada kepercayaan 95% = 1.96 𝛼: derajat kepercayaan 3.4 Data collection The population is an area that already consists of objects or
subjects with some capacities with specific characteristics 641 𝑛=
1.962 × 0.5(1 −0.5) × 10,000
0.052(10,000 −1) × 1.962 × 0.5(1 −0.5)
𝑛= 369.98
(2) 3.5 Wifi environment 𝑃: maksimal estimasi = 0.5 𝑑: alpha (0.05) atau sampling error = 5% 𝑑: alpha (0.05) atau sampling error = 5% Wifi measurements were taken at Building A at Campus 3
Unika Atma Jaya (at BSD) using the speed test application tool
to get the results from Figure 3. The result of the sample calculation using Eq. (1) by
inputting a population value of 10,000 can be calculated as
follows: ollows:
(a) AG floor
(b) 1st floor
(c) 2nd floor
(d) 3rd floor
(e) 4th floor
(f) 5th floor
(g) 6th floor
(h) 7th floor
Figure 3. Wi-Fi speedtest Figure 3. Wi-Fi speedtest 642 Wi-Fi dBm measurement
Wi-Fi Analyzer is a software that can measure the signal
ngth received through a cellphone so that it can see the
wer of the signal being tested by looking at the signal quality
l scale by Table 9. n the lake area (5.0 GHz access point). It can be seen from
Figure 4 that if the atma-student Wi-Fi has a signal strength
of -93 dBm, then the lake area signal strength is fair. In the canteen area (5.0 GHz access point). It can be
from the Figure 5 that if the atma-student Wi-Fi has a s
strength of -93 dBm, then the signal strength of the lake
is fair. In the park area (5.0 GHz access point). It can be seen
the Figure 6 that if the atma-student Wi-Fi has a signal stre
of -73 dBm, then the park area signal strength is good. (a) dBm
(b) Area
Figure 4. dBm of lake
(a) dBm
(b) Area
Figure 5. dBm of cafetaria of -93 dBm, then the lake area signal strength is fair. 3.6 Wi-Fi dBm measurement g
g
In the canteen area (5.0 GHz access point). It can be seen
from the Figure 5 that if the atma-student Wi-Fi has a signal
strength of -93 dBm, then the signal strength of the lake area
is fair. Wi-Fi Analyzer is a software that can measure the signal
strength received through a cellphone so that it can see the
power of the signal being tested by looking at the signal quality
level scale by Table 9. In the park area (5.0 GHz access point). It can be seen from
the Figure 6 that if the atma-student Wi-Fi has a signal strength
of -73 dBm, then the park area signal strength is good. In the lake area (5.0 GHz access point). It can be seen from
the Figure 4 that if the atma-student Wi-Fi has a signal strength (b) Area (b) Area (a) dBm (a) dBm
(b) Area
Figure 4. dBm of lake
(a) dBm
(b) Area
Figure 5. dBm of cafetaria
(a) dBm
(b) Area
Figure 6. dBm of park (b) Area (b) Area (a) dBm (a) dBm Figure 4. dBm of lake Figure 4. dBm of lake
(a) dBm
(b) Area
Figure 5. dBm of cafetaria (b) Area (a) dBm (b) Area Figure 5. dBm of cafetaria (a) dBm
(b) Area
Figure 6. dBm of park (b) Area (b) Area (b) Area Figure 6. dBm of park 643 Table 9. dBm Table
Signal Strength
RSSI
Excellent
> -70 dBm
Good
-70 dBm to -85 dBm
Fair
-86 dBm to -100 dBm
Poor
< -100 dBm
No Signal
-110 dBm Table 9. dBm Table Based on Figure 7, from the sample that has been taken,
Figure 7(a) 372 respondents for the faculty grouping each
respondent, the most samples who filled out the questionnaire
are the Faculty of Economics and Business as much as 24%,
the Faculty of Medicine as much as 26%, the Faculty of
Engineering as much as 18%, the Faculty of Technology as
much as 9%, the Faculty of Administrative Sciences and
Communication Sciences as much as 8%, the Faculty of Law
6%, the Faculty of Psychology 5%, and the last is the Faculty
of Education & Languages as much as 4%. Then Figure 7(b)
of the sample in terms of gender, the overall selection is male
55% & female 45%. Figure 7(c) is divided into 3 groupings of
campus areas from each respondent. 4.1 Analysis of respondents 4.1 Analysis of respondents .1 Analysis of respondents
(a) Respondent's faculty
(b) Respondent's gender
(c) Respondent's region
Figure 7. Respondents Figure 8. Normality test (a) Respondent's faculty (a) Respondent's faculty Figure 8. Normality test (b) Respondent's gender 4. RESULT AND DISCUSSION The following are the research instruments used in
collecting data. Researchers will use 98 statements based on
the Servqual dimension and the Likert Scale to measure each
piece of information presented to respondents. The design of
the statements in the questionnaire is taken from the indicators
determined in Servqual. In this study, researchers used 5
variables (X) consisting of Tangible (X1), Reliability (X2),
Responsiveness (X3), Assurance (X4), Empathy (X5) and 6
indicators of the variable (Y). So the number of indicators
obtained is 98 statements. 3.6 Wi-Fi dBm measurement The most samples filling
out the questionnaire are the Semanggi area, as much as 45%,
then the Bumi Serpong Damai (BSD) area, as much as 30%
and after that, the Pluit area, as much as 25%. Signal Strength
RSSI
Excellent
> -70 dBm
Good
-70 dBm to -85 dBm
Fair
-86 dBm to -100 dBm
Poor
< -100 dBm
No Signal
-110 dBm Signal Strength
RSSI
Excellent
> -70 dBm
Good
-70 dBm to -85 dBm
Fair
-86 dBm to -100 dBm
Poor
< -100 dBm
No Signal
-110 dBm 4.3 Validity test The validity test is conducted to help ensure that the
measuring instrument indeed measures what is intended or
accurately gauges, so that the results can be reliable and
relevant [37]. After testing the validity using SPSS 26.0, all
statements given to respondents are declared valid, and data
processing can be carried out. The following is a validity test
of each piece of information in the questionnaire for each
dimension: (1) Tangibles: range of person correlation 0,211 – 0,590
with R Table 0,10, the tangibles variable is said to be Valid. (b) Respondent's gender (2) Reliability: range of person correlation 0,201 – 0,607
with R Table 0,10, the Reliability variable is said to be Valid. (c) Respondent's region
Fi
7 R
d (3) Responsiveness: range of person correlation 0,260 –
0,576 with R Table 0,10, the Responsiveness variable is said
to be Valid. (4) Assurance: range of person correlation 0,307 – 0,556
with R Table 0,10, the Assurance variable is said to be Valid. (5) Empathy: range of person correlation 0,374 – 0,651 with
R Table 0,10, the Empathy variable is said to be Valid. Table 0,10, the Empathy variable is said to be Valid. (6) Satisfaction: range of person correlation 0,493 – 0,615
with R Table 0,10, the Satisfaction variable is said to be Valid. (c) Respondent's region 4.2 Normality test The sig value of Kolmogorv-Smirnov with the Monte Carlo
approach is 0,217, which means that the confidence level is
greater than 0,05 (0,217 > 0,05), and it is concluded that this
sample data shows the normal distribution [36] (see Figure 8). 4.5 Multicollinearity test Figure 11. ANOVA test result The multicollinearity test looks at the Tolerance Value and
VIF (Variance Inflation Factor). The trick is knowing whether
or not there is a multicollinearity test deviation by looking at
the Tolerance value > 0,10 and the VIF value < 10 [40]. If later
the multicollinearity test results are declared not to occur, the
predictive power is stable, and vice versa. If the data
processing results turn out to be multicollinearity, the
predictive power may not be reliable, so later, it may also not
be stable. Multicollinearity test result can been seen on Table
11. Figure 11. ANOVA test result Based on the results of the SPSS output above, it is known
that the R Square value is 0.522. It can be concluded that
52.2% of the variables Tangibles (X1), Reliability (X2),
Responsiveness (X3), Assurance (X4), and Empathy (X5) can
explain changes in Satisfaction (Y) and the remaining 47.8%
which they think is influenced by other factors outside the
variables that have been studied (see Figure 12). Table 11. Multicollinearity test result
Variable
Tolerance
VIF
Description
Tangibles
0,3220
3,1030
No Multicollinearity
Reliability
0,1500
6,6560
No Multicollinearity
Responsiveness
0,1680
5,9490
No Multicollinearity
Assurance
0,1430
6,9710
No Multicollinearity
Empathy
0,2010
4,9790
No Multicollinearity
4.6 Heteroscedasticity test Table 11. Multicollinearity test result Figure 12. Determination test result
Table 12 Hypothesis test result 4.6 Heteroscedasticity test Suppose the correlation between the independent variable
and the residual reaches a significance greater than 0.05 (Sig >
0,05). In that case, there is no heteroscedasticity problem, and
from the 5 variables above the sig value> 0.05, it is stated that
there is no heteroscedasticity (see Figure 9). Figure 12. Determination test result Table 12. Hypothesis test result Figure 9. Heteroscadistity test result
4.7 Multiple linear regression test
From the coefficients test results above, it is known that the Variable
Coefficient Value
T Statistics
Sig. Tangibles (β)
0.195
4,197
0,000
Reliability (β)
0.212
3,323
0,001
Responsiveness (β)
0.078
1,150
0,251
Assurance (β)
-0.297
-3,542
0,000
Empathy (β)
0.009
0,102
0,919
F-value
27.487 (1) Based on the coefficient test conducted and hypothesis
test result from Table 12, the regression coefficient (β) for the
Tangibles (X1) variable in relation to Satisfaction (Y) is 0.195. The obtained F-value is 27.487. Moreover, the Tangibles
variable demonstrates a significance value of 0.000, which is
lower than 0.05 (0.000 < 0.05). This indicates a significant
relationship between the variables examined in this study. The Figure 9. Heteroscadistity test result 4.4 Reliability test Figure 7. Respondents Figure 7. Respondents Reliability
testing
evaluates
the
constancy
and 644 regression coefficient (β) of the six variables on Tangibles is
0.194, Reliability 0.211, Responsiveness 0.079, Assurance -
0.296, Empathy 0.008 (see Figure 10). dependability of measurements or instruments employed in
research. This process guarantees that the measurements
produce dependable results consistently, both over time and
across various conditions [38]. This study uses Cronbach's
Alpha coefficient formula. Variables can be declared reliable
if they have a Cronbach's Alpha value > 0.60, and the results
obtained from the 6 variables above that the Cronbach alpha
value > 0.60, so the reliability test for the 6 variables above is
declared reliable [39]. Reliability test result can be seen on
Table 10. Figure 10. Coefficients test result Table 10. Reliability test result
Variable
Variable
Description
Tangibles
0,60
Reliable
Reliability
0,60
Reliable
Responsiveness
0,60
Reliable
Assurance
0,60
Reliable
Empathy
0,60
Reliable
Satisfaction
0,60
Reliable
4.5 Multicollinearity test Table 10. Reliability test result Figure 10. Coefficients test result Based on the significance test table above, the Sig. Value is
0.000 < 0.05 and Fcount > Ftable is 27,486 > 2.27, then Ho is
rejected, so it can be stated that there is a significant influence
between Tangibles (X1), Reliability (X2), Responsiveness
(X3), Assurance (X4), Empathy (X5) on Satisfaction (Y) (see
Figure 11). 4.5 Multicollinearity test 5. CONCLUSIONS This research addresses the issue of user satisfaction
evaluation with Information Technology (IT) services at Atma
Jaya Catholic University (Unika Atma Jaya), with a specific
focus on the dimensions of service quality. The approach used
in this study is the Service Quality (Servqual) model,
successfully assessing user satisfaction with IT services at
Unika Atma Jaya using the variables Tangibles (physical
evidence), Reliability, Responsiveness, Assurance, and
Empathy. (3) According to the aforementioned coefficient test, the
regression coefficient (β) for the Responsiveness (X3) variable
in relation to Satisfaction (Y) is 0.078. The obtained F-value
is 27.487. However, the significance value for the
Responsiveness variable is 0.251, which exceeds 0.05 (0.251 >
0.05). This indicates an absence of a significant relationship
between the variables examined in this study. The T-value is
1.150, smaller than the T-table value of 1.97 (1.150 < 1.97). Hence, the hypothesis is rejected, signifying that there is no
influence between Responsiveness (X3) and Satisfaction (Y). Observing that Unika Atma Jaya has provided clear Standard
Operating Procedures (SOP) regarding constraints in
accessing applications and facilities, where users must follow
the specified procedures. This is what causes Responsiveness
not to have an impact on Satisfaction due to timing issues. (3) According to the aforementioned coefficient test, the
regression coefficient (β) for the Responsiveness (X3) variable
in relation to Satisfaction (Y) is 0.078. The obtained F-value
is 27.487. However, the significance value for the
Responsiveness variable is 0.251, which exceeds 0.05 (0.251 >
0.05). This indicates an absence of a significant relationship
between the variables examined in this study. The T-value is
1.150, smaller than the T-table value of 1.97 (1.150 < 1.97). Hence, the hypothesis is rejected, signifying that there is no
influence between Responsiveness (X3) and Satisfaction (Y). Observing that Unika Atma Jaya has provided clear Standard
Operating Procedures (SOP) regarding constraints in
accessing applications and facilities, where users must follow
the specified procedures. This is what causes Responsiveness
not to have an impact on Satisfaction due to timing issues. p
y
Upon a comprehensive examination of the test results, it
becomes evident that certain dimensions of service quality
have varying impacts on user satisfaction within the Unika
Atma Jaya environment. Particularly, the Tangibles and
Reliability dimensions emerge as significant contributors,
indicating a positive and statistically significant impact on user
satisfaction. 4.7 Multiple linear regression test From the coefficients test results above, it is known that the From the coefficients test results above, it is known that the From the coefficients test results above, it is known that the 645 T-value is 4.197, exceeding the T-table value of 1.97 (4.197 >
1.97), thereby leading to the acceptance of the hypothesis. Therefore, it can be concluded that Tangibles (X1) have an
influence on Satisfaction (Y). This can be interpreted as some
applications of Unika Atma Jaya do not yet have a sufficiently
good user interface design and fail to provide clear information
on app usage. Additionally, the lack of adequate computer
facilities and WiFi accessibility for all users contributes to the
impact on satisfaction. T-value is 4.197, exceeding the T-table value of 1.97 (4.197 >
1.97), thereby leading to the acceptance of the hypothesis. Therefore, it can be concluded that Tangibles (X1) have an
influence on Satisfaction (Y). This can be interpreted as some
applications of Unika Atma Jaya do not yet have a sufficiently
good user interface design and fail to provide clear information
on app usage. Additionally, the lack of adequate computer
facilities and WiFi accessibility for all users contributes to the
impact on satisfaction. and facilities through email. This information typically does
not have a high level of urgency and is not a common problem,
so it does not affect Satisfaction. The calculation results indicate that user satisfaction with IT
services at Unika Atma Jaya show that the Tangibles and
Reliability variables have a significant positive impact on user
satisfaction, while the Responsiveness variable does not have
a significant influence. Moreover, the Assurance variable
shows a significant negative impact, whereas the Empathy
variable does not demonstrate a significant influence on user
satisfaction. (2) In reference to the coefficient test results, the regression
coefficient (β) for the Reliability (X2) variable with respect to
Satisfaction (Y) is 0.212. The obtained F-value is 27.487. Furthermore, the Reliability variable exhibits a significance
value of 0.001, which is less than 0.05 (0.001 < 0.05). Consequently, the variables used in this study display a
significant relationship. The T-value is 3.323, surpassing the
T-table value of 1.97 (3.323 > 1.97), leading to the acceptance
of the hypothesis. Thus, it can be concluded that Reliability
(X2) influences Satisfaction (Y). It can be concluded that some
applications of Unika Atma Jaya are unable to provide
accurate and reliable information. 4.7 Multiple linear regression test Furthermore, computer
facilities and Wifi accessibility should be easily accessible at
any time. These factors have implications for user satisfaction. 5. CONCLUSIONS This is evident from the significant values of
Tangibles and Reliability variables, which are 0.000 and 0.001,
respectively, with T-table values of 4.197 and 3.323. This
suggests that improvements or enhancements in tangible
aspects of service, such as facilities and physical evidence, as
well as reliability in service delivery, can substantially
increase
overall
user
satisfaction. Conversely,
the
Responsiveness dimension, although tested, does not show a
statistically significant impact on user satisfaction as it has a
significant value of 0.251 and a T-table value of 1.150. This
indicates that users' perceptions of prompt service delivery
may not be a determining factor in their overall satisfaction. Interestingly, the Assurance dimension, while having the
potential to build trust, demonstrates a significant negative
impact on user satisfaction. The Assurance variable has a
significant value of 0.000 and a T-table value of -3.542. This
implies that careful management of aspects related to
assurance is crucial to avoid negative effects on overall user
satisfaction. Additionally, the Empathy dimension does not
show a significant impact on user satisfaction with a
significant value of 0.919 and a T-table value of 0.102. This
indicates that empathetic understanding of user needs may not
be a major factor in shaping user satisfaction in this context. (4) Based on the provided coefficient test, the regression
coefficient (β) for the Assurance variable (X4) in relation to
Satisfaction (Y) is -0.297. The obtained F-value is 27.487. For
the Assurance variable, the significance value is 0.000, which
is less than 0.05 (0.000 < 0.05). This indicates a significant
relationship between the variables examined in this study. The
calculated T-value is -3.542, exceeding the T-table value of
1.97 (-3.542 > 1.97). Therefore, the hypothesis is rejected,
suggesting that there is no influence between Assurance (X4)
and Satisfaction (Y). This can be seen in the fact that Unika
Atma Jaya's application has provided services according to
user needs and instilled a level of confidence in user data
protection rights. Therefore, this can be interpreted as
Assurance having a negative impact on Satisfaction. (4) Based on the provided coefficient test, the regression
coefficient (β) for the Assurance variable (X4) in relation to
Satisfaction (Y) is -0.297. The obtained F-value is 27.487. For
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no influence between Empathy (X5) and Satisfaction (Y). Usually, IT support provides service-related information
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directions. Focus could be placed on the Assurance dimension,
which indicates negative impacts, with in-depth research to
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https://openalex.org/W2495795714
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https://europepmc.org/articles/pmc5295397?pdf=render
|
English
| null |
RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors
|
Oncotarget
| 2,016
|
cc-by
| 11,849
|
1Department of Clinical Neuroendocrinology, Max Planck Institute of Psychiatry, Munich, Germany 1Department of Clinical Neuroendocrinology, Max Planck Institute of Psychiatry, Munich, Germany 2Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society,
Buenos Aires, Argentina 2Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society,
Buenos Aires, Argentina 2Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of
Buenos Aires, Argentina 3Institute of Pathology, Technical University of Munich, Munich, Germany 3Institute of Pathology, Technical University of Munich, Munich, Germany 4Department of Internal Medicine II, University-Hospital Campus Grosshadern, Interdisciplinary Center of Neuroendocrine
Tumours of the GastroEnteroPancreatic System (GEPNET-KUM), Ludwig-Maximilians-University of Munich, Munich, Germany 5Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, Buenos Aires, Argentina 5Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, Buenos Aires, Argentina 6Current address: German Cancer Research Center, Heidelberg, Germany 7Current address: Institute of Pathology, University of Düsseldorf, Düsseldorf, Germany Correspondence to: Ulrich Renner, email: renner@mpipsykl.mpg.de
Keywords: RSUME, RWDD3, PanNETs, angiogenesis, metastasis
Received: November 04, 2015 Accepted: July 17, 2016 Published: August 05, 2016 Published: August 05, 2016 Abstract The factors triggering pancreatic neuroendocrine tumor (PanNET) progression are
largely unknown. Here we investigated the role and mechanisms of the sumoylation
enhancing protein RSUME in PanNET tumorigenesis. Immunohistochemical studies
showed that RSUME is strongly expressed in normal human pancreas, in particular
in β-cells. RSUME expression is reduced in insulinomas and is nearly absent in other
types of PanNETs suggesting a role in PanNET tumorigenesis. In human pancreatic
neuroendocrine BON1 cells, RSUME stimulates hypoxia-inducible factor-1α (HIF-1α)
and vascular endothelial growth factor-A (VEGF-A), which are key components of tumor
neovascularisation. In contrast, RSUME suppresses nuclear factor-κB (NF-κB) and its
target interleukin-8 (IL-8). Correspondingly, PanNET cells with RSUME knockdown
showed decreased HIF-1α activity and increased NF-κB and IL-8 production leading
to a moderate reduction of VEGF-A release as reduced HIF-1α/VEGF-A production is
partly compensated by NF-κB/IL-8-induced VEGF-A. Notably, RSUME stabilizes the
tumor suppressor PTEN, which is frequently lost in PanNETs and whose absence is
associated with metastasis formation. In vivo orthotopic transplantation of PanNET
cells with or without RSUME expression into nude mice showed that PanNETs without
RSUME have reduced PTEN expression, grow faster and form multiple liver metastases. In sum, RSUME differentially regulates key components of PanNET formation suggesting
that the observed loss of RSUME in advanced PanNETs is critically involved in PanNET
tumorigenesis, particularly in metastasis formation. www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget/ Yonghe Wu1,6, Lucas Tedesco2, Kristin Lucia1, Anna M. Schlitter3, Jose Monteserin Garcia1,
Irene Esposito3,7, Christoph J. Auernhammer4, Marily Theodoropoulou1, Eduardo Arzt2,5,
Ulrich Renner1, Günter K. Stalla1 Yonghe Wu1,6, Lucas Tedesco2, Kristin Lucia1, Anna M. Schlitter3, Jose Monteserin Garcia1,
Irene Esposito3,7, Christoph J. Auernhammer4, Marily Theodoropoulou1, Eduardo Arzt2,5,
Ulrich Renner1, Günter K. Stalla1 partment of Clinical Neuroendocrinology, Max Planck Institute of Psychiatry, Munich, Germany INTRODUCTION as insulinomas, glucagonomas, gastrinomas, etc. [1, 2]. About 50 to 60% of the tumors are functionally active
and are diagnosed according to their symptoms caused
by the hormonal hypersecretion [2, 3]. Functionally
inactive PanNETs, if not accidentally detected, are mostly
recognized in advanced stages when they have formed
large invasive tumors and distant metastases [1, 2, 4]. Pancreatic neuroendocrine tumors (PanNETs)
are rare and represent about 1 to 3% of all neoplasias
of the pancreas [1, 2]. They derive from endocrine cells
producing pancreatic hormones such as insulin, glucagon,
gastrin, and others, and are correspondingly designated www.impactjournals.com/oncotarget Oncotarget 57878 According to the WHO classification of 2010, all PanNETs
are considered to be potentially malignant [1, 4]. Thus, the
majority shows a malignant phenotype forming metastases
in neighboring tissue or lymph nodes and almost
40–50% have hepatic metastasis at diagnosis [1, 4]. As an
exception, the majority of insulinomas appear as clinically
benign and only approximately 10 % of all insulinomas
are malignant [1, 2, 4]. The cumulative 5-year survival
rate of patients with PanNETs has been reported to be
83%, which drops to around 27% in advanced malignant
stages with high Ki-67 index and the formation of distant
metastases [1, 2, 4]. under normoxic conditions. Under hypoxia, as it occurs in
expanding tumors, this process is rapidly inhibited leading
to the production of active an HIF-1 complex, which then
induces the production of multiple angiogenic factors
including VEGF-A [16]. We have previously shown that RSUME, the
product of the RWDD3 gene, is a RWD containing
protein that stabilizes and enhances HIF-1α [17–19]. RSUME also stabilizes I-κBα, the natural inhibitor of
NF-κB, thereby inhibiting NF-κB [17, 19]. The latter
is overexpressed in many types of tumors, including
pancreatic adenocarcinomas, where it plays a key
role in mediating inflammatory signals and triggering
proliferation, angiogenesis and metastasis [20]. One of the
NF-κB targets, interleukin-8 (IL-8), is strongly expressed
in PanNETs, suggesting NF-κB over-activation [21]. Since pancreas strongly expresses RSUME [17], which
is involved in the regulation of key molecules and
processes influencing PanNETs, the aim of the study
was to explore the expression and role of RSUME in
PanNET tumorigenesis. We found that loss of RSUME is
a characteristic of PanNETs and may contribute to tumor
angiogenesis and metastasis. RSUME is down-regulated in human pancreatic
neuroendocrine tumors PanNETs are in some cases associated with
hereditary
genetic
syndromes
such
as
multiple
endocrine neoplasia type I (MEN1), von Hippel-Lindau
(VHL) syndrome, tuberous sclerosis syndrome or von
Recklinghausen´s disease [1, 2, 9]. Inactivating somatic
mutations of several genes have also been reported in
PanNETs, in particular in the tumor suppressor MEN1,
in the chromatin interaction proteins (death-domain
associated protein) DAXX/ (alpha thalassemia/mental
retardation syndrome X-linked) ATRX, and in negative
regulators of the PI3K-Akt-mTOR signaling cascade,
e.g. phosphatase and tensin homolog (PTEN) or tuberous
sclerosis 2 (TSC2) [1, 2, 10–12]. Loss of function of
the tumor suppressor gene PTEN is frequently found in
PanNETs and is responsible for the over-activation of the
PI3K-Akt-mTOR cascade [13, 14]. Immunohistochemical studies showed strong
RSUME expression in the insulin-positive cells of
the Langerhans islets in the normal pancreas (n = 9)
(Figure 1A, 1E), in which somatostatin-positive cells also
expressed RSUME (Supplementary Figure 1). Moderate
expression of RSUME was also found in exocrine
acinar cells whereas RSUME was absent in ductal cells
(Figure 1B–1E). Among 24 islet 1-positive PanNETs
[22] investigated (11 G1 and 13 G2 tumors; Table 1,
Supplementary Figure 2), scattered cytoplasmatic RSUME
immunopositivity was observed in insulinomas (n = 7;
Figure 1C, 1E) whereas RSUME was absent in the vast
majority of the other PanNETs including 4 somatostatin
expressing tumors (Figure 1B, 1D, 1E; Supplementary
Figure 1). Thus, in comparison to the normal pancreas,
RSUME expression is decreased in PanNETs (Figure 1F). PanNETs are densely vascularized and express
HIF-1α and VEGF-A [7, 15]. In contrast to many types
of tumors, in which loss of differentiation goes along with
an increase of microvessel density, less well-differentiated
PanNETs are paradoxically less vascularized probably
due to an impaired expression of the HIF-1/VEGF-A
pathway [15]. VEGF-A is regulated by HIF-1, which plays
a key role in tumor neovascularization and is composed
of the constitutively expressed HIF-1β and the oxygen-
sensitive HIF-1α subunit [16]. The latter is continuously
produced but subsequently degraded by ubiquitinylation INTRODUCTION Complete resection of the primary tumors is the
only curative therapeutic approach, however due to the
formation of distant liver metastasis at diagnosis resection
is only possible in a low percentage of patients [5, 6]. Palliative therapeutic options for inoperable pancreatic
neuroendocrine tumors include treatment with somatostatin
analogues, peptide receptor radionuclide therapy and
chemotherapy using streptozocin- or temozolomide-based
protocols [5, 6]. Due to the high degree of vascularization
of PanNETs, the efficacy of anti-angiogenic acting drugs
has also been approved [7]. Moreover, drugs targeting
various receptor kinases or components of intracellular
signaling pathways have been tested in recent clinical
trials [7, 8]. However, the overall prognosis is still not
satisfactory and better understanding of the molecular
mechanisms of PanNETs development is urgent need in
order to develop improved therapeutic approaches. RSUME regulates angiogenesis through the
HIF-1α/VEGF pathway In neuroendocrine pancreatic BON1 cells, RSUME
mRNA and protein was enhanced by hypoxia (1% O2;
Figure 2A) or hypoxia mimicking conditions (CoCl2
treatment, Supplementary Figure 3) through RSUME/
HIF-1 interaction (Supplementary Figure 4) as already
shown in other cell types [17, 23]. Knocking down
RSUME (BON1RSUME-KD) decreased basal and hypoxia- www.impactjournals.com/oncotarget Oncotarget 57879 during hypoxia confirming the impact of RSUME on
HIF-1α regulation (Supplementary Figure 5).i induced RSUME and HIF-1α mRNA and protein levels
(Figure 2B, 2C), confirming the importance of RSUME in
HIF-1α regulation. In comparison to the strong suppression
of moderately inhibited basal and hypoxia-induced mRNA
levels and secretion of VEGF-A (Figure 2D) suggesting
the induction of a compensatory mechanism to preserve
VEGF-A production. In neuroendocrine pancreatic QGP1
cells, RSUME overexpression induced HIF-1a expression In HIF-1α deficient colon cancer cells, VEGF-A
production is preserved by the pro-angiogenic cytokine
IL-8 [24]. We found expression of IL-8 and its receptor
CXCR2 in BON1 cells and in the human neuroendocrine
carcinoma QGP1 cell line (Supplementary Figure 6). The
CXCR2 inhibitor SB225002 significantly reduced basal Figure 1: RSUME expression is decreased in human pancreatic neuroendocrine tumors. Immunohistochemistry stainin
of RSUME in resected normal pancreas (A), PanNETs (B, Grade 2), insulinoma (C) and PanNET with a non-malignant normal region
(D, Grade 1). (E) Co-staining of Insulin (green) and RSUME (red) in normal pancreas, insulinoma, and other types of PanNETs. Image
are representative of three experiments with similar results. Scale bar 50 µm. (F) Summary of RSUME expression in normal pancreas
insulinoma and other types of PanNETs. The intensity of the staining was classified as negative (0), weakly (1+), medium (2+) and strongly
positive (3+). All samples from this study were assessed by two different raters who were blinded to each other. See Table 1 for detaile
patient information. Figure 1: RSUME expression is decreased in human pancreatic neuroendocrine tumors. Immunohistochemistry staining
of RSUME in resected normal pancreas (A), PanNETs (B, Grade 2), insulinoma (C) and PanNET with a non-malignant normal region
(D, Grade 1). (E) Co-staining of Insulin (green) and RSUME (red) in normal pancreas, insulinoma, and other types of PanNETs. Images
are representative of three experiments with similar results. Scale bar 50 µm. (F) Summary of RSUME expression in normal pancreas,
insulinoma and other types of PanNETs. RSUME regulates angiogenesis through the
HIF-1α/VEGF pathway The intensity of the staining was classified as negative (0), weakly (1+), medium (2+) and strongly Figure 1: RSUME expression is decreased in human pancreatic neuroendocrine tumors. Immunohistochemistry staining
of RSUME in resected normal pancreas (A), PanNETs (B, Grade 2), insulinoma (C) and PanNET with a non-malignant normal region
(D, Grade 1). (E) Co-staining of Insulin (green) and RSUME (red) in normal pancreas, insulinoma, and other types of PanNETs. Images
are representative of three experiments with similar results. Scale bar 50 µm. (F) Summary of RSUME expression in normal pancreas,
insulinoma and other types of PanNETs. The intensity of the staining was classified as negative (0), weakly (1+), medium (2+) and strongly
positive (3+). All samples from this study were assessed by two different raters who were blinded to each other. See Table 1 for detailed
patient information. www.impactjournals.com/oncotarget This effect was abolished when
I-κBα was mutated at the SUMO1 conjunction target sites
lysines 21 and 22 (Figure 3B, right, lane 1 and 2) [26]
or overexpression of the RSUME-Mut (Y61A, P62A) and hypoxia-induced VEGF-A secretion (Supplementary
Figure
7). RSUME
knockdown
increased
IL-8
transcription and secretion, which was further induced
by hypoxia (Figure 2E). Increased levels of IL-8 can
stimulate VEGF-A, which may explain that the loss of
RSUME in PanNET cells has limited inhibitory effects on
VEGF-A secretion despite strongly decreased HIF-1α. and hypoxia-induced VEGF-A secretion (Supplementary
Figure
7). RSUME
knockdown
increased
IL-8
transcription and secretion, which was further induced
by hypoxia (Figure 2E). Increased levels of IL-8 can
stimulate VEGF-A, which may explain that the loss of
RSUME in PanNET cells has limited inhibitory effects on
VEGF-A secretion despite strongly decreased HIF-1α. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 57880 Table 1: Clinicopathological features of PanNET patients
Diagnosis
Grading
Proliferation
index
Hormone
Expression
RSUME
expression
PTEN
level
PTEN
subloc. Islet1
1
PanNET
G1
3%
Not tested
−
−
+++
2
PanNET
G1
< 1%
Not tested
−
++
N + C
+++
3
PanNET,
lymphnode
metastasis
G2
15%
No expression
−
++
C
+++
4
Insulinoma
G1
3%
Insulin+
++
++
C
+++
5
PanNET
G2
NA
Not tested
−
+++
6
PanNET
G2
NA
Insulin neg. ++
+
C
+++
7
PanNET
G1
1%
Not tested
−
−
+++
8
PanNET
G2
4%
Not tested
−
−
+++
9
PanNET
G2
NA
Serotonin+,
Somatostatin+
Gastrin+
−
+
C
+++
10
PanNET
G2
3%
Not tested
−
+++
C
+
11
PanNET
G2
10%
Somatostatin+
−
−
+
12
PanNET
G2
7%
Insulin neg. +
−
++
13
PanNET
G1
< 2%
Insulin neg. −
++
C
++
14
PanNET
G2
15%
No expression
−
−
+++
15
PanNET
G2
3%
Somatostatin+
−
−
+++
16
PanNET,
lymphnode
metastasis
G1
1%
Glucagon+
−
++
C
++
17
PanNET,
lymphnode
metastasis
G1
< 1%
Somatostatin+ VIP
−
+
N + C
+++
18
PanNET
G2
4%
Serotonin+, Insulin
neg. ++
−
+++
19
Insulinoma
G2
NA
Insulin+
+
−
+++
20
Insulinoma
G1
NA
Insulin+
+++
+
C
−
21
Insulinoma,
liver
metastasis
G2
5%
Insulin+
+
−
+++
22
Insulinoma
G1
< 2%
Insulin+
+++
−
+++
23
Insulinoma
G2
3%
Insulin+
+
++
C
+++
24
Insulinoma
G1
NA
Insulin+
+++
++
C
+++
PanNET: pancreatic neuroendocrine tumor; NA: not available; N: Nuclear staining; C: cytoplasm staining; subloc:
sublocalization. Table 1: Clinicopathological features of PanNET patients PanNET: pancreatic neuroendocrine tumor; NA: not available; N: Nuclear staining; C: cytoplasm staining; subloc:
sublocalization. promoter activity and co-transfection with the I-κBα
super repressor (I-κBα-SR) significantly attenuated this
effect (Figure 3A, left). All these effects were completely
abolished when the NF-κB binding site of the IL-8
promoter was mutated, which further demonstrates
that RSUME inhibits IL-8 activity through NF-κB in
BON1 cells (Figure 3A, right). RSUME overexpression
increased I-κBα sumoylation, an effect which was
comparable to that of SUMO1 (Figure 3B, left, upper
band, lanes 2 and 4). RSUME negatively regulates NF-κB activity by
enhancing IκBα sumoylation in PanNETs IL-8 expression is stimulated by NF-κB [25]. RSUME overexpression inhibited TNFa-induced IL-8 www.impactjournals.com/oncotarget Oncotarget 57881 p65/RelA and I-κBα. RSUME knockdown increased
phosphorylated I-κBα (Ser32/36) and p65/RelA (Ser536)
levels (Figure 3E), indicating NF-κB activation. Furthermore, loss of RSUME is accompanied by increased
p65/RelA (Ser536) nuclear translocation (Figure 3F),
which is indicative of activated NF-κB. where the highly conserved YPXXXP motif in the RWD
domain of RSUME was mutated (Figure 3B, right, lane 3
and 4) [17, 22]. Co-transfection with the SUMO1/sentrin
specific peptidase 1 (SENP1), attenuated sumoylated
I-κBα (Figure 3B, left, lanes 3, 5, 6) demonstrating
that RSUME specifically affects I-κBα sumoylation. RSUME suppressed basal and TNFa-induced NF-κB
transcriptional activity similar to SUMO1, and this effect
was abolished by the I-κBα super-repressor (Figure 3C). In contrast, RSUME knockdown increased both basal
and TNFa-induced NF-κB transcriptional activity
(Figure 3D), further demonstrating the repressive role of
RSUME on NF-κB activity in BON1 cells. RSUME enhances SUMO-directed PTEN
sumoylation (B) BON1
cells were co-transfected with I-κBα, I-κBα mutated at the SUMO1 conjunction target sites lysine 21 and 22 (K21, 22R), and the indicated
expression vectors (including the SUMO1/sentrin specific peptidase 1 -SENP1), to analyze I-κBα sumoylation status in BON1 cells. After
24 h, cell extracts were subjected to WB with anti-I-κBα antibody. β-actin was used as the loading control. (C) BON1 cells were transfected
with NF-κB-LUC reporter vector, RSUME, HA-SUMO1 or I-κBα expression vectors. After 24 h, cells were stimulated with 10 ng/ml
TNF-α for 6 h, and LUC activity was measured. (D) NF-κB-Luc activity in BON1RSUME-KD and BON1Scramble cells was measured with or
without TNF-α (10 ng/ml, 6 h). All values were normalized to β-gal activity (mean ± SEM of 3 different experiments, t test). *P < 0.05,
**P < 0.01 (E) Western blot experiments were performed with cell extracts were RSUME was knockdown and with Immunoblotting for
NF-κB subunits (p65, p-p65(Ser536), I-κBα and pI-κBα (Ser32/36) in BON wild type, BON1RSUME-KD and BON1Scramble cells lysates. β-actin
was used as the loading control. (F) Immunofluorescence for total and phosphor-p65 (Ser536) in BON1RSUME-KD and BON1Scramble cells. Antibodies used were indicated above each image with corresponding colors. DAPI staining was used to visualize the nucleus. Scale bars
are equal to 10 μm. Each image is representative of three independent experiments with similar results. Figure 3: RSUME negatively regulates NF-κB activity by enhancing sumoylation of IκBα. BON1 cells were transfected
with IL-8-LUC (A, left) or IL-8 (NF-κB-mut)-LUC (A, right) reporter vector, RSUME or IκBα super repressor (I-κBα-SR) and β-gal
plasmid. After 24 h, cells were stimulated with 10 ng/ml TNF-α for 6 h, and LUC activity was measured in the cell extracts. (B) BON1
cells were co-transfected with I-κBα, I-κBα mutated at the SUMO1 conjunction target sites lysine 21 and 22 (K21, 22R), and the indicated
expression vectors (including the SUMO1/sentrin specific peptidase 1 -SENP1), to analyze I-κBα sumoylation status in BON1 cells. After
24 h, cell extracts were subjected to WB with anti-I-κBα antibody. β-actin was used as the loading control. (C) BON1 cells were transfected
with NF-κB-LUC reporter vector, RSUME, HA-SUMO1 or I-κBα expression vectors. After 24 h, cells were stimulated with 10 ng/ml
TNF-α for 6 h, and LUC activity was measured. (D) NF-κB-Luc activity in BON1RSUME-KD and BON1Scramble cells was measured with or
without TNF-α (10 ng/ml, 6 h). RSUME enhances SUMO-directed PTEN
sumoylation NF-κB was previously shown to suppress PTEN
[27], which is reduced in 16 of 24 of the PanNETs
studied (Table 1). Since RSUME negatively regulates
NF-κB activity, we investigated its role on PTEN
transcription. RSUME overexpression in BON1 cells
increased PTEN mRNA and protein levels and the Western blot experiments were performed with
cell extracts using antibodies against key components of
the NF-κB pathway, including total and phosphorylated O
57882
i
tj
l
/
t
t
Figure 2: Influence of RSUME on HIF-1α, VEGF-A and IL-8 production. RSUME mRNA and protein level (A) were
stimulated during hypoxia (1% O2) for the indicated time points in BON1 cells. As expected RSUME mRNA and protein was down-
regulated and less sensitive to hypoxia in BON1 cells with RSUME knock-down (BON1RSUME-KD) compared to scramble siRNA trasfected
cells (BON1Scramble) (B). HIF-1a mRNA and in particular hypoxia-induced HIF-1a protein production was strongly impaired in BON1RSUME-KD
cells (C). Normoxic and hypoxic mRNA synthesis and secretion of VEGF-A was suppressed in BON1RSUME-KD cells (D) whereas IL-8
mRNA and protein production was enhanced. (E). All experiments were performed three times and in B to E treatment time was 3 h for
mRNA expression and 12 h for protein production studies, respectively. Results are expressed as mean ± SEM of triplicates for mRNA and
quadruplicate for ELISA. *P < 0.05, **P < 0.01. Figure 2: Influence of RSUME on HIF-1α, VEGF-A and IL-8 production. RSUME mRNA and protein level (A) were
stimulated during hypoxia (1% O2) for the indicated time points in BON1 cells. As expected RSUME mRNA and protein was down-
regulated and less sensitive to hypoxia in BON1 cells with RSUME knock-down (BON1RSUME-KD) compared to scramble siRNA trasfected
cells (BON1Scramble) (B). HIF-1a mRNA and in particular hypoxia-induced HIF-1a protein production was strongly impaired in BON1RSUME-KD
cells (C). Normoxic and hypoxic mRNA synthesis and secretion of VEGF-A was suppressed in BON1RSUME-KD cells (D) whereas IL-8
mRNA and protein production was enhanced. (E). All experiments were performed three times and in B to E treatment time was 3 h for
mRNA expression and 12 h for protein production studies, respectively. Results are expressed as mean ± SEM of triplicates for mRNA and
quadruplicate for ELISA. *P < 0.05, **P < 0.01. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 57882 3: RSUME negatively regulates NF-κB activity by enhancing sumoylation of IκBα. RSUME enhances SUMO-directed PTEN
sumoylation BON1 cells we
-8-LUC (A, left) or IL-8 (NF-κB-mut)-LUC (A, right) reporter vector, RSUME or IκBα super repressor (I-κBα-S
. After 24 h, cells were stimulated with 10 ng/ml TNF-α for 6 h, and LUC activity was measured in the cell extrac
re co-transfected with I-κBα, I-κBα mutated at the SUMO1 conjunction target sites lysine 21 and 22 (K21, 22R), and
on vectors (including the SUMO1/sentrin specific peptidase 1 -SENP1), to analyze I-κBα sumoylation status in BON
l extracts were subjected to WB with anti-I-κBα antibody. β-actin was used as the loading control. (C) BON1 cells we
F-κB-LUC reporter vector, RSUME, HA-SUMO1 or I-κBα expression vectors. After 24 h, cells were stimulated w
for 6 h, and LUC activity was measured. (D) NF-κB-Luc activity in BON1RSUME-KD and BON1Scramble cells was mea
TNF-α (10 ng/ml, 6 h). All values were normalized to β-gal activity (mean ± SEM of 3 different experiments, t tes
01 (E) Western blot experiments were performed with cell extracts were RSUME was knockdown and with Immu RSUME negatively regulates NF-κB activity by enhancing sumoylation of IκBα. BON1 cells we
UC (A, left) or IL-8 (NF-κB-mut)-LUC (A, right) reporter vector, RSUME or IκBα super repressor (I-κBα-
er 24 h, cells were stimulated with 10 ng/ml TNF-α for 6 h, and LUC activity was measured in the cell extra
o-transfected with I-κBα, I-κBα mutated at the SUMO1 conjunction target sites lysine 21 and 22 (K21, 22R), an
ectors (including the SUMO1/sentrin specific peptidase 1 -SENP1), to analyze I-κBα sumoylation status in BO
racts were subjected to WB with anti-I-κBα antibody. β-actin was used as the loading control. (C) BON1 cells w
-LUC reporter vector, RSUME, HA-SUMO1 or I-κBα expression vectors. After 24 h, cells were stimulated
h and LUC activity was measured (D) NF-κB-Luc activity in BON1RSUME-KD and BON1Scramble cells was me Figure 3: RSUME negatively regulates NF-κB activity by enhancing sumoylation of IκBα. BON1 cells were transfected
with IL-8-LUC (A, left) or IL-8 (NF-κB-mut)-LUC (A, right) reporter vector, RSUME or IκBα super repressor (I-κBα-SR) and β-gal
plasmid. After 24 h, cells were stimulated with 10 ng/ml TNF-α for 6 h, and LUC activity was measured in the cell extracts. RSUME increases PTEN nuclear accumulation PTEN nuclear localization is pivotal for its anti-
tumorigenic activity and is regulated by sumoylation
[28, 29]. PTEN displayed differential sublocalization
between the normal pancreas and PanNETs (Figure 6A,
Table 1), similar to previous observation [30]. In
the normal pancreas, PTEN had strong nuclear and
cytoplasmic staining in insulin-producing islet cells
(Figure 6A, left), whereas in PanNET samples, PTEN
showed either loss of expression (Figure 6A, right) or
predominantly cytoplasmic staining (Figure 6A, middle). Wild type GFP-PTEN was equally distributed in both
the nucleus and cytoplasm while the PTEN mutants
(K254R, K266R, K254/266R) showed predominantly
cytoplasmic localization (Figure 6B, left). RSUME
overexpression (red) dramatically increased nuclear
PTEN accumulation (Figure 6B, right). Similar results
were obtained in HEK293 and PTEN-null prostate PC3
cells (Supplementary Figure 10). RSUME overexpression
had only moderate effects on the nuclear translocation
of the single PTEN-K254R and PTEN-K266R mutants
but clearly reduced nuclear translocation double PTEN-
K254/266R mutant, which remained predominantly in
the cytoplasm (Figure 6B, Supplementary Figure 10). In
sum, RSUME facilitates PTEN nuclear accumulation by
modifying its sumoylation status. RSUME enhances SUMO-directed PTEN
sumoylation All values were normalized to β-gal activity (mean ± SEM of 3 different experiments, t test). *P < 0.05,
**P < 0.01 (E) Western blot experiments were performed with cell extracts were RSUME was knockdown and with Immunoblotting for
NF-κB subunits (p65, p-p65(Ser536), I-κBα and pI-κBα (Ser32/36) in BON wild type, BON1RSUME-KD and BON1Scramble cells lysates. β-actin
was used as the loading control. (F) Immunofluorescence for total and phosphor-p65 (Ser536) in BON1RSUME-KD and BON1Scramble cells. Antibodies used were indicated above each image with corresponding colors. DAPI staining was used to visualize the nucleus. Scale bars
are equal to 10 μm. Each image is representative of three independent experiments with similar results. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 57883 opposite was observed after RSUME knockdown
(Figure 4A). In QGP1 cells RSUME overexpression also
enhanced PTEN expression (Supplementary Figure 5). The functional significance of RSUME´s effect on PTEN
is evidenced by the increased Akt phosphorylation in
RSUME knockdown cells (Supplementary Figure 8). In
addition, we observed a high migration band (~75 kDa,
H-PTEN), which is regulated by RSUME, indicating
posttranslational modification (Supplementary Figure 8). RSUME knockdown decreased total and 75kDa
H-PTEN: total PTEN ratio (13.13 ± 4.77% vs. 5.97 ±
1.95%, Supplementary Figure 8). We confirmed that
the higher migration band at ~75 kDa is SUMO-PTEN
by co-immunoprecipitation with SUMO2 (Figure 4B). An in vitro sumoylation assay revealed that the band
detected between 75 and 100 kDa (~26 kDa for GST-tag)
was SUMO2-modified PTEN, and this signal was
further enhanced by RSUME (Figure 4C). A SUMO
conjugation assay confirmed the increase in 75 kDa
sumoylated PTEN levels in RSUME overexpressing
cells (Figure 4D, lane 3). RSUME also enhanced
SUMO1-mediated PTEN sumoylation, but to a lesser
extent (Supplementary Figure 9). To further clarify the
mechanisms through which RSUME regulates PTEN we
used COS7 cells as we assume that the effects of RSUME
on PTEN may be similar in each RSUME expressing cell
type. RSUME-Mut (Y61A, P62A) failed to stimulate
PTEN modification by SUMO2 (Figure 4D, lane 4). In silico prediction (SUMOplot™ Analysis Program) and
previous reports [28, 29] revealed two major sumoylation
conjugation sites (lysine 254 and lysine 266) in PTEN. Sumoylation deficient PTEN mutants (K254R, K266R,
K254/266R) displayed variable sumoylation (Figure 4E,
lanes 2–4). The expected ~75 kDa was observed in Ni-
NTA purified proteins when cells were co-transfected
with wild type PTEN (WT-PTEN) and His-SUMO2. RSUME enhances SUMO-directed PTEN
sumoylation To further clarify the
mechanisms through which RSUME regulates PTEN we
used COS7 cells as we assume that the effects of RSUME
on PTEN may be similar in each RSUME expressing cell
type. RSUME-Mut (Y61A, P62A) failed to stimulate
PTEN modification by SUMO2 (Figure 4D, lane 4). In silico prediction (SUMOplot™ Analysis Program) and
previous reports [28, 29] revealed two major sumoylation
conjugation sites (lysine 254 and lysine 266) in PTEN. Sumoylation deficient PTEN mutants (K254R, K266R,
K254/266R) displayed variable sumoylation (Figure 4E,
lanes 2–4). The expected ~75 kDa was observed in Ni-
NTA purified proteins when cells were co-transfected
with wild type PTEN (WT-PTEN) and His-SUMO2. The single K254R mutant showed reduced sumoylation
(Figure 4E, lane 2 vs. lane 1), which was further reduced
in the double K254/266R mutant (lane 4). In contrast,
the single K266R mutant (lane 3) had a minor effect on
sumoylation, but exerts a synergistic effect with the K254
site. RSUME overexpression strongly enhanced SUMO2-
induced sumoylation in the WT-PTEN construct (lane 5),
but not in the double K254/266R mutant (lane 8). Co-
immunoprecipitation revealed a physical association
between RSUME and PTEN in cells (Figure 4F). RSUME knockdown in BON1 cells showed
decreased PTEN half-life during cycloheximide (CHX)
treatment while it had little effect in the scramble
transfected control cells (Figure 5D). Similarly, the
sumoylation deficient PTEN-K254/266R displayed
decreased protein half-life compared to WT-PTEN
indicating the critical role of sumoylation for PTEN
protein stabilization. RSUME overexpression significantly
increased WT-PTEN protein stability, but it had little
effect on protein stability of the double mutant K254/266R
(Figure 5E). RSUME increases PTEN stability by reducing its
ubiquitination We next investigated if the RSUME-induced PTEN
sumoylation modulates PTEN protein stability. RSUME
overexpression strongly reduced PTEN ubiquitination
levels in COS7 cells, indicative of enhanced protein
stability (Figure 5A). The inhibitory role of RSUME www.impactjournals.com/oncotarget RSUME enhances SUMO-directed PTEN
sumoylation The single K254R mutant showed reduced sumoylation
(Figure 4E, lane 2 vs. lane 1), which was further reduced
in the double K254/266R mutant (lane 4). In contrast,
the single K266R mutant (lane 3) had a minor effect on
sumoylation, but exerts a synergistic effect with the K254
site. RSUME overexpression strongly enhanced SUMO2-
induced sumoylation in the WT-PTEN construct (lane 5),
but not in the double K254/266R mutant (lane 8). Co-
immunoprecipitation revealed a physical association
between RSUME and PTEN in cells (Figure 4F). on PTEN ubiquitination was further confirmed by
silencing RSUME in COS7 cells (Figure 5B). PTEN-
K254R and the double mutant K254/266R displayed
increased ubiquitination compared to wild type, while
the single K266R mutation had a minor effect on PTEN
ubiquitination. RSUME overexpression inhibited PTEN
ubiquitination in the wild type as well as single mutants
(K254R and K266R), suggesting that a single mutation at
the sumoylation acceptor site is not sufficient to prevent
RSUME-induced PTEN ubiquitination inhibition. When
both PTEN sumoylation acceptor sites were mutated,
RSUME overexpression had no inhibitory effect on PTEN
ubiquitination. These results indicate that RSUME inhibits
PTEN ubiquitination and that both sumoylation acceptor
sites (lysine 254 and lysine 266) are indispensable for
sustaining this effect (Figure 5C). opposite was observed after RSUME knockdown
(Figure 4A). In QGP1 cells RSUME overexpression also
enhanced PTEN expression (Supplementary Figure 5). The functional significance of RSUME´s effect on PTEN
is evidenced by the increased Akt phosphorylation in
RSUME knockdown cells (Supplementary Figure 8). In
addition, we observed a high migration band (~75 kDa,
H-PTEN), which is regulated by RSUME, indicating
posttranslational modification (Supplementary Figure 8). RSUME knockdown decreased total and 75kDa
H-PTEN: total PTEN ratio (13.13 ± 4.77% vs. 5.97 ±
1.95%, Supplementary Figure 8). We confirmed that
the higher migration band at ~75 kDa is SUMO-PTEN
by co-immunoprecipitation with SUMO2 (Figure 4B). An in vitro sumoylation assay revealed that the band
detected between 75 and 100 kDa (~26 kDa for GST-tag)
was SUMO2-modified PTEN, and this signal was
further enhanced by RSUME (Figure 4C). A SUMO
conjugation assay confirmed the increase in 75 kDa
sumoylated PTEN levels in RSUME overexpressing
cells (Figure 4D, lane 3). RSUME also enhanced
SUMO1-mediated PTEN sumoylation, but to a lesser
extent (Supplementary Figure 9). www.impactjournals.com/oncotarget Oncotarget 57884 RSUME knockdown alters tumorigenic factors
and favors metastasis formation in an orthotopic
tumor model tumors in the pancreas by 9 weeks and the tumors from
the RSUME-KD group showed much higher weight
compared to scramble controls (1102.5 ± 78.5 versus 786
± 56.2 mg, Supplementary Figure 12). BON1RSUME-KD tumor
samples exhibited significantly higher Ki67 proliferation
index (12.70 ± 1.07% in BON1RSUME-KD tumors versus
10.15 ± 1.01% in BON1Scramble, P < 0.01) (Supplementary
Figure 12), which is in line with the proliferation results
in vitro (Supplementary Figure 11). BON1RSUME-KD displayed
reduced chromogranin A (CgA) immunoreactivity
compared to the scramble control (Figure 7A) indicating Our data show that loss of RSUME upregulates
NF-κB and downregulates PTEN, and both these effects
may contribute to enhanced PanNET tumorigenesis. We made orthotopic xenografts by implanting human
pancreatic BON1 PanNET (scramble and RSUME-KD)
cells into the pancreas of nude mice, which was proven to
be a successful PanNET model [31]. All animals developed igure 4: RSUME enhances SUMO-induced PTEN sumoylation. (A) PTEN mRNA expression was measured in RSU
verexpressing (right) and RSUME silenced (left) BON1 cells. β-actin was used for normalization. Immunoblot for PTEN was analy
n RSUME silenced (left) and RSUME overexpressing (right) BON1 cells. β-actin was used for normalization. High-migration PT
H-PTEN). (B) Immunoprecipitation was performed with a PTEN antibody in BON1 cells followed by western blot using antibo
gainst PTEN and SUMO2. (C) GST-PTEN was incubated using an in vitro sumoylation assay mixture containing SUMO2 with or wit
SUME. After incubation, reactions were stopped by adding loading buffer and subsequently an immunoblot was performed with
TEN antibody. (D) COS7 cells were co-transfected with HA-PTEN, wild type V5-RSUME or the mutated V5-RSUME (Y61A,P6
is6-SUMO2 and V5-Ubc9. 48 h post-transfection, cells were lysed, purified by Ni-NTA and immunoblotted with indicated antibod
E) COS7 cells were co-transfected with HA-PTEN expression or sumoylation-deficient PTEN mutants (K254R, K266R, K254/26
is6-SUMO2 and V5-RSUME plasmids. 48 h post-transfection, cells were lysed, Ni-NTA purified and immunoblotted with the indic
ntibodies (F) COS7 cells were transfected with HA PTEN with or without V5 RSUME 48 h post transfection cells were lysed Figure 4: RSUME enhances SUMO-induced PTEN sumoylation. (A) PTEN mRNA expression was measured in RSUM
verexpressing (right) and RSUME silenced (left) BON1 cells. β-actin was used for normalization. Immunoblot for PTEN was analyze
n RSUME silenced (left) and RSUME overexpressing (right) BON1 cells. β-actin was used for normalization. High-migration PTEN
H-PTEN). RSUME knockdown alters tumorigenic factors
and favors metastasis formation in an orthotopic
tumor model (B) Immunoprecipitation was performed with a PTEN antibody in BON1 cells followed by western blot using antibodie
gainst PTEN and SUMO2. (C) GST-PTEN was incubated using an in vitro sumoylation assay mixture containing SUMO2 with or withou
RSUME. After incubation, reactions were stopped by adding loading buffer and subsequently an immunoblot was performed with ant
TEN antibody. (D) COS7 cells were co-transfected with HA-PTEN, wild type V5-RSUME or the mutated V5-RSUME (Y61A,P62A
His6-SUMO2 and V5-Ubc9. 48 h post-transfection, cells were lysed, purified by Ni-NTA and immunoblotted with indicated antibodie
E) COS7 cells were co-transfected with HA-PTEN expression or sumoylation-deficient PTEN mutants (K254R, K266R, K254/266R
His6 SUMO2 and V5 RSUME plasmids 48 h post transfection cells were lysed Ni NTA purified and immunoblotted with the indicate Figure 4: RSUME enhances SUMO-induced PTEN sumoylation. (A) PTEN mRNA expression was measured in RSUME
overexpressing (right) and RSUME silenced (left) BON1 cells. β-actin was used for normalization. Immunoblot for PTEN was analyzed
in RSUME silenced (left) and RSUME overexpressing (right) BON1 cells. β-actin was used for normalization. High-migration PTEN
(H-PTEN). (B) Immunoprecipitation was performed with a PTEN antibody in BON1 cells followed by western blot using antibodies
against PTEN and SUMO2. (C) GST-PTEN was incubated using an in vitro sumoylation assay mixture containing SUMO2 with or without
RSUME. After incubation, reactions were stopped by adding loading buffer and subsequently an immunoblot was performed with anti-
PTEN antibody. (D) COS7 cells were co-transfected with HA-PTEN, wild type V5-RSUME or the mutated V5-RSUME (Y61A,P62A),
His6-SUMO2 and V5-Ubc9. 48 h post-transfection, cells were lysed, purified by Ni-NTA and immunoblotted with indicated antibodies. (E) COS7 cells were co-transfected with HA-PTEN expression or sumoylation-deficient PTEN mutants (K254R, K266R, K254/266R),
His6-SUMO2 and V5-RSUME plasmids. 48 h post-transfection, cells were lysed, Ni-NTA purified and immunoblotted with the indicated
antibodies. (F) COS7 cells were transfected with HA-PTEN with or without V5-RSUME. 48 h post-transfection, cells were lysed with
RIPA buffer (10% for Input), precipitated with PTEN antibody and subsequently immunoblot was performed with antibodies indicated. For
all experiments, one representative experiment from two independent experiments with similar results is shown. Figure 4: RSUME enhances SUMO-induced PTEN sumoylation. (A) PTEN mRNA expression was measured in RSUME
overexpressing (right) and RSUME silenced (left) BON1 cells. β-actin was used for normalization. Immunoblot for PTEN was analyzed
in RSUME silenced (left) and RSUME overexpressing (right) BON1 cells. β-actin was used for normalization. High-migration PTEN
(H-PTEN). RSUME knockdown alters tumorigenic factors
and favors metastasis formation in an orthotopic
tumor model (B) Immunoprecipitation was performed with a PTEN antibody in BON1 cells followed by western blot using antibodies
against PTEN and SUMO2. (C) GST-PTEN was incubated using an in vitro sumoylation assay mixture containing SUMO2 with or without
RSUME. After incubation, reactions were stopped by adding loading buffer and subsequently an immunoblot was performed with anti-
PTEN antibody. (D) COS7 cells were co-transfected with HA-PTEN, wild type V5-RSUME or the mutated V5-RSUME (Y61A,P62A),
His6-SUMO2 and V5-Ubc9. 48 h post-transfection, cells were lysed, purified by Ni-NTA and immunoblotted with indicated antibodies. (E) COS7 cells were co-transfected with HA-PTEN expression or sumoylation-deficient PTEN mutants (K254R, K266R, K254/266R),
His6-SUMO2 and V5-RSUME plasmids. 48 h post-transfection, cells were lysed, Ni-NTA purified and immunoblotted with the indicated
antibodies. (F) COS7 cells were transfected with HA-PTEN with or without V5-RSUME. 48 h post-transfection, cells were lysed with
RIPA buffer (10% for Input), precipitated with PTEN antibody and subsequently immunoblot was performed with antibodies indicated. For
all experiments, one representative experiment from two independent experiments with similar results is shown. www.impactjournals.com/oncotarget Oncotarget 57885 pp65/RelA-Ser536
and
the
NF-kB
target
IL-8
(Supplementary Figures 13C, 14, 16), confirming our in
vitro data. In addition, IL-8 transcripts were elevated in the
BON1RSUME-KD tumors (Supplementary Figure 13C). In
contrast, PTEN expression was significantly reduced in
BON1RSUME-KD xenografts with predominantly cytoplasmic
staining (Supplementary Figures 13D, 14, 17). BON1Scramble
tumors showed strong expression (mostly nuclear) of
PTEN, despite its pancreatic neuroendocrine tumor origin
(Supplementary Figures 13D, 14, 17). Accordingly,
activated pAkt-Ser473 immunoreactivity and protein
levels were increased in BON1RSUME-KD tumors compared
to the scramble controls (Supplementary Figures 14, 18). loss of neuroendocrine differentiation. BON1RSUME-KD
derived tumors showed weak cytoplasmic HIF-1α
immunoreactivity compared to the strong nuclear staining
seen in BON1Scramble (Supplementary Figure 15). In addition,
BON1RSUME-KD derived tumors showed slightly lower VEGF
immunoreactivity (not shown) and mRNA expression
compared to BON1Scramble (Supplementary Figure 13B). In contrast, BON1RSUME-KD tumors showed lower micro-
vessel density (MVD) compared to scramble control
(44.8 ± 3.6/field versus 67.4 ± 6.3/field) as determined by
CD31 immunostaining (Figure 7B). BON1RSUME-KD
tumors
showed
stronger
immunoreactivity for the activated NF-kB subunit igure 5: RSUME inhibits PTEN ubiquitination and increases protein stability. (A) COS7 cells were transfected with
A-PTEN, His6-ubiquitin and V5-RSUME or a backbone construct pCEFL. 48 h post-transfection, cells were incubated with 5 µM
G132 or vehicle for 6 h. RSUME knockdown alters tumorigenic factors
and favors metastasis formation in an orthotopic
tumor model His-ubiquitinated (His-Ub) proteins were isolated from denatured whole-cell extracts and pulled down by nickel
ads. Purified proteins and input samples (whole-cell extracts) were analyzed by western blotting with anti-PTEN and anti-V5 (RSUME)
ntibodies. Signal in His-Ub pull-down lanes corresponds to ubiquitinated PTEN. b-actin was used as a loading control. (B) COS7 cells were
ansfected with HA-PTEN, His6-ubiquitin and 10 µM siRSUME. 48 h posttransfection, cells were incubated with MG132, purified and
mmunoblotted with anti-PTEN and anti-RSUME. β-actin was used as a loading control. (C) COS7 cells were transfected with HA-PTEN
different mutants (K254R, K266R, K254/266R), V5-RSUME and His6-Ubiquitin. 48 h post-transfection, cells were incubated with
G132, purified and immunoblotted with anti-PTEN and anti-RSUME. One representative experiment from two independent experiments
ith similar results is shown. (D) BON1RSUME-KD and BON1Scramble cells were treated with cycloheximide (CHX) for the time indicated. Cell
sates were collected and subjected to immunoblot using antibodies against PTEN and RSUME. β-actin was used as the loading control. E) COS7 cells were co-transfected with wild type or sumoylation-deficient PTEN mutant (K254/266R) with or without V5-RSUME. 48 h
ost-transfection, cells were treated with cycloheximide for the indicated time points and immunoblotted with the indicated antibodies. actin was used as the loading control. Figure 5: RSUME inhibits PTEN ubiquitination and increases protein stability. (A) COS7 c ts PTEN ubiquitination and increases protein stability. (A) COS7 cells were transfected with
d
b
kb
h
f
i
ll
i
b
d
i h Figure 5: RSUME inhibits PTEN ubiquitination and increases protein stability. (A) COS7 cells were transfected with
HA-PTEN, His6-ubiquitin and V5-RSUME or a backbone construct pCEFL. 48 h post-transfection, cells were incubated with 5 µM
MG132 or vehicle for 6 h. His-ubiquitinated (His-Ub) proteins were isolated from denatured whole-cell extracts and pulled down by nickel
beads. Purified proteins and input samples (whole-cell extracts) were analyzed by western blotting with anti-PTEN and anti-V5 (RSUME)
antibodies. Signal in His-Ub pull-down lanes corresponds to ubiquitinated PTEN. b-actin was used as a loading control. (B) COS7 cells were
transfected with HA-PTEN, His6-ubiquitin and 10 µM siRSUME. 48 h posttransfection, cells were incubated with MG132, purified and
immunoblotted with anti-PTEN and anti-RSUME. β-actin was used as a loading control. (C) COS7 cells were transfected with HA-PTEN
or different mutants (K254R, K266R, K254/266R), V5-RSUME and His6-Ubiquitin. 48 h post-transfection, cells were incubated with
MG132, purified and immunoblotted with anti-PTEN and anti-RSUME. DISCUSSION Five of eight (62.5%) mice injected with
BON1RSUME-KD cells developed liver metastasis, evidenced
grossly and histologically by H&E staining (Figure
7C, 7D). In contrast, only one out of eight (12.5%)
mice injected with BON1Scramble cells developed liver
metastasis. Markers for epithelial-mesenchymal transition
(EMT), such as TGFβ, Snail, N-cadherin increased, while
E-cadherin decreased in BON1RSUME-KD tumors, indicating
that loss of RSUME promotes EMT (Figure 7E). Altogether, our data show that loss of RSUME in PanNET
cells results in PTEN loss and supports PanNET tumor and
metastasis formation. In the present study, we demonstrate for the first
time that the expression of RSUME is altered in PanNETs
and present in vitro and in vivo data which strongly
suggest that the reduction of RSUME contributes to the
tumorigenesis and metastasis in PanNETs. RSUME is
highly expressed in various endocrine/exocrine glands,
including the pancreas [17]. We could show that RSUME
is mainly localized in insulin-producing beta-cells and is
also present in other endocrine and exocrine pancreatic
cell types whereas it is not expressed in the ductal cells, O
t
57887
w impactjournals com/oncotarget
igure 6: RSUME influences PTEN localization in normal and tumoral pancreatic cells. (A) Immunofluorescence studi
owed that in the normal pancreas, PTEN (red) is predominantly localized in the nuclei of insulin-producing cells (green) (A, le
hereas in PanNETs PTEN is present in the cytoplasm of tumor cells (A, middle) or completely absent (A, right). PTEN showed bo
uclear and cytoplasmic expression in BON1 cells transfected with GFP-PTEN (green) whereas the PTEN sumoylation deficient doub
utant (K254/266R) (green) is localized only in the cytoplasm (B, left). Over-expression of RSUME (red) in GFP- or K254/266R-PTE
xpressing BON1 cells strongly increased nuclear PTEN (green) expression in cells with GFP-PTEN whereas PTEN remained in t
ytoplasm of cells expressing the K254/266R mutant (B, right). Cell nuclei were visualized using DAPI (blue). Scale bar: 10 μm. Figure 6: RSUME influences PTEN localization in normal and tumoral pancreatic cells. (A) Immunofluorescence studies
showed that in the normal pancreas, PTEN (red) is predominantly localized in the nuclei of insulin-producing cells (green) (A, left)
whereas in PanNETs PTEN is present in the cytoplasm of tumor cells (A, middle) or completely absent (A, right). PTEN showed both
nuclear and cytoplasmic expression in BON1 cells transfected with GFP-PTEN (green) whereas the PTEN sumoylation deficient double
mutant (K254/266R) (green) is localized only in the cytoplasm (B, left). RSUME knockdown alters tumorigenic factors
and favors metastasis formation in an orthotopic
tumor model One representative experiment from two independent experiments
with similar results is shown. (D) BON1RSUME-KD and BON1Scramble cells were treated with cycloheximide (CHX) for the time indicated. Cell
lysates were collected and subjected to immunoblot using antibodies against PTEN and RSUME. β-actin was used as the loading control. (E) COS7 cells were co-transfected with wild type or sumoylation-deficient PTEN mutant (K254/266R) with or without V5-RSUME. 48 h
post-transfection, cells were treated with cycloheximide for the indicated time points and immunoblotted with the indicated antibodies. β-actin was used as the loading control. Figure 5: RSUME inhibits PTEN ubiquitination and increases protein stability. (A) COS7 cells were transfected with
HA-PTEN, His6-ubiquitin and V5-RSUME or a backbone construct pCEFL. 48 h post-transfection, cells were incubated with 5 µM
MG132 or vehicle for 6 h. His-ubiquitinated (His-Ub) proteins were isolated from denatured whole-cell extracts and pulled down by nickel
beads. Purified proteins and input samples (whole-cell extracts) were analyzed by western blotting with anti-PTEN and anti-V5 (RSUME)
antibodies. Signal in His-Ub pull-down lanes corresponds to ubiquitinated PTEN. b-actin was used as a loading control. (B) COS7 cells were
transfected with HA-PTEN, His6-ubiquitin and 10 µM siRSUME. 48 h posttransfection, cells were incubated with MG132, purified and
immunoblotted with anti-PTEN and anti-RSUME. β-actin was used as a loading control. (C) COS7 cells were transfected with HA-PTEN
or different mutants (K254R, K266R, K254/266R), V5-RSUME and His6-Ubiquitin. 48 h post-transfection, cells were incubated with
MG132, purified and immunoblotted with anti-PTEN and anti-RSUME. One representative experiment from two independent experiments
with similar results is shown. (D) BON1RSUME-KD and BON1Scramble cells were treated with cycloheximide (CHX) for the time indicated. Cell
lysates were collected and subjected to immunoblot using antibodies against PTEN and RSUME. β-actin was used as the loading control. (E) COS7 cells were co-transfected with wild type or sumoylation-deficient PTEN mutant (K254/266R) with or without V5-RSUME. 48 h
post-transfection, cells were treated with cycloheximide for the indicated time points and immunoblotted with the indicated antibodies. β-actin was used as the loading control. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 57886 DISCUSSION Over-expression of RSUME (red) in GFP- or K254/266R-PTEN
expressing BON1 cells strongly increased nuclear PTEN (green) expression in cells with GFP-PTEN whereas PTEN remained in the
cytoplasm of cells expressing the K254/266R mutant (B, right). Cell nuclei were visualized using DAPI (blue). Scale bar: 10 μm. www.impactjournals.com/oncotarget Oncotarget 57887 indicating a distinct physiological role of RSUME in
endocrine and exocrine functions of the pancreas. Whereas
RSUME expression was preserved in all insulinomas
studied, RSUME was mostly absent in other types of
hormone-producing and in hormone-negative PanNETs. As most of the insulinomas are considered to represent
well-differentiated, benign tumors we speculate that the
loss of RSUME is associated with the development of
more aggressive, less well-differentiated PanNETs. After
this first description on the role of RSUME in PanNETs,
clinical correlation studies are needed to establish the
association that RSUME might have whit different types
of tumors. in 2204 breast cancer patients RWDD3 was associated
with paclitaxel-induced neuropathy [34], which however
could not be confirmed in a cohort of paclitaxel-treated
patients with ovarian cancer [35]. Alterations of RSUME
have also been associated with chronic inflammation-
induced neuropathic pain [36]. Hypoxia up-regulated RSUME in the PanNET-
derived BON1 cell line, but in PanNETs RSUME
was down-regulated suggesting the presence of other
more important regulatory mechanisms. Inflammatory
processes play an important role in the development of
pancreatic adenocarcinomas [2] and may be responsible
for
the
RSUME
down-regulation
in
PanNETs. Indeed, TNF-a which was reported to be produced
intratumorally in PanNETs [36], strongly suppressed
RSUME (Supplementary Figure 19). These finding
are in accordance with a recent study in which chronic
inflammation reduced the transcription of RWDD3/
RSUME and anti-inflammatory acting drugs could revert
this effect [36]. Tumor expansion is accompanied by transient
hypoxia, which triggers tumor neovascularization. Hypoxia induces RSUME, which was found to be
upregulated in the hypoxic inner zones of gliomas and
pituitary adenomas [17, 32]. In breast cancer, RSUME
(RWDD3) has been associated with 15 other genes as part
of the risk prediction signature [33]. In a GWAS performed Figure 7: Role of RSUME in an orthotopic neuroendocrine pancreatic tumor model. Orthotopic tumors from BON1 cells
without (scramble) or with RSUME knockdown (RSUME-KD) were generated by injecting the cells into the pancreas of athymic nude
mice. In comparison to scramble tumors, RSUME-KD tumors showed reduced chromogranin A staining (brown) indicative of a loss of
neuroendocrine differentiation (A). DISCUSSION RSUME-KD tumors are significantly less vascularised (CD31 staining) than scramble tumors (B)
but show strongly enhanced spread of metastases into the liver as shown morphologically (C) and immunohistochemically by detecting
CgA-positive neuroendocrine tumor tissue in the nude mouse liver (D). Tumors with RSUME-KD show reduced PTEN expression and
signs of epithelial-mesenchymal-transition (EMT) such as enhanced TGF-β, N-Cadherin and Snail protein levels as well as reduced
E-Cadherin protein expression (E) indicating that loss of RSUME promotes EMT in PanNETs. Microvessel density in the right panel in
B was determined by counting of the number of vessels in one field under 100× magnification. Results were obtained from 6 independent
pictures for each condition and are expressed as mean ± SEM. *P < 0.05 vs. scramble tumors. Scale bar 100 µm. Figure 7: Role of RSUME in an orthotopic neuroendocrine pancreatic tumor model. Orthotopic tumors from BON1 cells
without (scramble) or with RSUME knockdown (RSUME-KD) were generated by injecting the cells into the pancreas of athymic nude
mice. In comparison to scramble tumors, RSUME-KD tumors showed reduced chromogranin A staining (brown) indicative of a loss of
neuroendocrine differentiation (A). RSUME-KD tumors are significantly less vascularised (CD31 staining) than scramble tumors (B)
but show strongly enhanced spread of metastases into the liver as shown morphologically (C) and immunohistochemically by detecting
CgA-positive neuroendocrine tumor tissue in the nude mouse liver (D). Tumors with RSUME-KD show reduced PTEN expression and
signs of epithelial-mesenchymal-transition (EMT) such as enhanced TGF-β, N-Cadherin and Snail protein levels as well as reduced
E-Cadherin protein expression (E) indicating that loss of RSUME promotes EMT in PanNETs. Microvessel density in the right panel in
B was determined by counting of the number of vessels in one field under 100× magnification. Results were obtained from 6 independent
pictures for each condition and are expressed as mean ± SEM. *P < 0.05 vs. scramble tumors. Scale bar 100 µm. www.impactjournals.com/oncotarget Oncotarget 57888 Loss of RSUME abolished the stimulatory effect
of hypoxia on HIF-1α, but not on VEGF-A, suggesting
the presence of an alternative compensatory mechanism. We have previously shown that RSUME inhibits NF-κB
by stabilizing its inhibitor I-κBα [17]. NF-κB stimulates
VEGF-A transcription directly by binding its promoter
or indirectly through IL-8 expression, which acts in an
autocrine fashion [21, 24]. DISCUSSION Our data show that loss of
RSUME induces IL-8 synthesis, which then triggers
VEGF-A, an effect that is abolished using an IL-8 receptor
CXCR2 inhibitor. Poorly differentiated PanNETs are
less vascularized than well-differentiated tumors [15]. Furthermore, high expression of IL-8 and CXCR2 was
observed in the human PanNET patient samples [21]. Thus, the NF-κB/IL-8 pathway may compensate
the decline of HIF-1 action on VEGF-A-mediated
angiogenesis in PanNET cells that have lost RSUME. This may explain why neovascularization is maintained
in advanced PanNETs despite RSUME down-regulation. Herein, we demonstrate that loss of RSUME results in high
metastatic potential in orthotopic pancreatic transplants. We
have chosen the orthotopic transplantation in the pancreas
compared to subcutaneous transplantation, because it mimics
the human PanNET condition and the metastasis formation
of the tumors in the neighboring structures of the pancreas,
in particular in the liver, can be monitored [30, 43]. Tumors
of BON1 cells with RSUME knockdown formed multiple
liver metastases that may be a consequence of increased
NF-κB expression and/or loss of PTEN [12, 30, 44]. The
BON1RSUME-KD-derived tumors were also much larger,
had higher proliferation rates and signs of high-grade less
well-differentiated tumors as shown by morphological
H&E staining and decreased CgA expression. In addition,
microvessel density was low in these tumors similar to what
is observed in the human PanNETs [15]. Our data demonstrate that RSUME affects multiple
targets in PanNET cells and as long as RSUME expression
is preserved, these tumors show relatively low proliferation
rates, expand slowly and have a limited metastatic
potential despite elevated angiogenesis and enhanced
microvessel density. The decline of RSUME in PanNETs
goes along with reduced HIF-1α/VEGF-A, elevated
NF-κB/IL-8, declined PTEN and enhanced PI3K/Akt/
mTOR activation. These multiple changes may explain
why mono-targeting pharmacological treatment concepts
often failed in tumor therapy and therefore combined
application of drugs directed against different targets gave
better results [5–8]. Altogether, our findings demonstrate
considerable evidence that loss of RSUME is involved
in the increase of tumor aggressiveness and metastases
formation in PanNETs. After our first description on the
involment of RSUME in PanNET, further studies with
higher numbers of PanNETs are needed in which the
correlations between RSUME expression and histological
characteristics of the tumors as well as clinical parameters
and outcome of affected patients are investigated. Another important target, which is inhibited by NF-κB
is PTEN [27]. DISCUSSION PTEN is reduced in PanNETs and its
expression is inversely correlated with survival [13, 14]. PTEN loss is rarely caused by genetic mutations in
PanNETs and the mechanism remains unclear [11, 30]. In
the present study, we describe for the first time that the
tumor suppressor PTEN is a target of RSUME. Loss of
RSUME in a PanNET model in nude mice is accompanied
by a decrease in PTEN, which may be in part due to the
upregulation of NF-κB that suppresses PTEN transcription
[27]. In addition, recent studies demonstrated that PTEN
is sumoylated [28, 38] and herein, we show that RSUME
enhances PTEN sumoylation and its activity. This action
on PTEN reinforces the role of RSUME on several
specific targets related to cancer and inflammation, such as
HIF-1a, I-κB, GR and pVHL [17, 18, 23, 32, 39]. Whether
the observed loss of RSUME in the majority of human
PanNETs studied is responsible for the cytoplasmatic
localization or complete loss of PTEN in our series of
human PanNETs needs to be confirmed in future studies. Human tissue samples Paraffin-embedded tissue slides of 9 normal
pancreas
tissue
samples,
24
human
pancreatic
neuroendocrine tumors and intraoperatively removed
adjacent normal human pancreatic tissue were obtained
from the Department of Pathology of the Technical
University Munich, Germany. The histopathological
diagnosis and grading, as well as staging followed the
recommendations of the WHO (Table 1). Sampling of
tissues and usage of clinical data for scientific purposes
was approved by the institutional ethics committee. MATERIALS AND METHODS Loss of RSUME is accompanied by decreased PTEN
sumoylation and increased Akt phosphorylation, which may
contribute to the PI3K pathway over-activation observed
in high grade PanNETs [11, 14]. The decreased PTEN
sumoylation impairs its cellular distribution. Cytosolic
PTEN suppress the PI3K survival pathway, while nuclear
PTEN controls DNA damage repair, genotoxic stress,
chromosome stabilization and growth [29, 40, 41]. Our
data show that RSUME sumoylates PTEN and increases
its nuclear accumulation. Indeed, sumoylated PTEN is
located in the nucleus, where it induces DNA repair upon
genotoxic stress [29, 40]. Therefore, the observed RSUME
down-regulation leads to loss of nuclear PTEN, which
would result in impaired chromosome stabilization [40, 41]. Immunoprecipitation Cells for immunoprecipitation assay were lysed in
Immunoprecipitation lysis buffer (25 mM Tris PH 8.0,
150 mM NaCl, 1% NP-40, 1 mM EDTA, 20 mM NEM)
containing protease inhibitor cocktail (1:100, Sigma). The
whole cell lysates obtained from centrifugation were first
precleared by Dynabeads Protein G magnetic (Invitrogen)
for 1 hour and then incubated with specific antibodies
(PTEN, # 9558, Cell Signaling, 1:100) overnight at 4°C
with rotation. The protein G magnetic beads were added
the following day to the lysates and further incubate for
2 hours at 4°C with rotation. The immunocomplexes were
then washed with Immunoprecipitation lysis buffer three
times and then boiled with sample loading buffer and
subjected to SDS-PAGE followed by western blot analysis. Cell lines and reagents Increased chromosomal instability renders the
tumor cell susceptible to additional mutations that increase
its tumorigenic and metastatic potential [2, 13, 14, 42]. Human pancreatic endocrine tumor BON1 cells
used in the study were authenticated by Eurofins www.impactjournals.com/oncotarget Oncotarget 57889 (Ebersberg, Germany). HEK293 cells and COS7 cells
were obtained from American Type Culture Collection
(ATCC; Manassas, VA, USA) and were cultured - like
BON1 cells - at 5% CO2 and 37°C in DMEM (pH 7.3)
supplemented with 10% FCS, 2mM glutamine, 0.5 mg/l
partricin and 105 U/liter penicillin-streptomycin. In
addition to BON1 cells, human pancreatic endocrine
carcinoma QGP-1 cells were used for several control
experiments as some authors suggest that QGP1 cells were
a better model for PanNETs. However, in a recent study
only limited differences between the two cell types were
found [45]. QGP1 cells were obtained from the Health
Science Research Resources Bank (Osaka, Japan) and
maintained in RPMI1640 medium supplemented with 10%
FCS, 2mM glutamine, 0.5 mg/l partricin and 105 U/liter
penicillin-streptomycin. Cell culture materials and
reagents were obtained from Life Technologies, Falcon
(Heidelberg, Germany), Nunc (Wiesbaden, Germany),
Seromed (Berlin, Germany), Flow Cytometry Standards
Corp. (Meckenheim, Germany) and Sigma. Cobalt
chloride (CoCl2) was purchased from Sigma. For hypoxia,
cells were incubated in 2% serum at 37°C, 5% CO2, and
1% O2 balanced with N2 using a hypoxic chamber ProOx
Model 110 (BioSpherix). For stable RSUME knockdown generation, the plasmids
encoding shRSUME or scramble RNA (SABiosciences,
USA) were transfected with lipofectamin 2000 into the
BON1 cells according to the standard protocol. The stable
clones were selected with Geneticin (Life Technologies)
at a concentration of 1 mg/ml. After the selection, stable
clones with RSUME knockdown (BON1RSUME-KD) and
scramble (BON1Scramble) were cultured in medium with
500 µg/ml Geneticin and used at passage 4 and 5. RSUME
expression level was validated by RT-PCR and western
blot. All the data shown were from one individual clone
designated No.15; similar results were obtained in other
individual clones as well as in transient expression assays. Western blot and antibodies Western blot analysis was carried out on whole cell
extracts (50 µg) upon various treatments, after fractionation
by PAGE gel electrophoresis and transferred to PVDF
membranes for immunoblotting with antibodies listed
in Supplementary Table 3. HRP-conjugated secondary
antibodies against rabbit and mouse were all obtained
from Cell Signalling Tech. The ECL system (Clarity ECL
substrate, Biorad) and hyperfilm (GE Healthcare, Munich,
Germany) were used for membrane visualization. Transfection and generation of target-specific
silent cells All constructs used in this study are listed in
Supplementary Table 2. Sumoylation deficient PTEN
mutations (K254R, K266R, K254/266R) were generated
by site-directed mutagenesis assay as described previously. Transfection assay was performed with lipofectamin 2000
following the manufacturer’s instructions. The reporter
constructs (HRE-Luc, RSUME-Luc, RSUME-∆HRE-Luc,
IL-8-Luc, IL-8-∆-NFκB-Luc) were described previously. 500 ng of reporter constructs with 300 ng β-galactosidase
were co-transfected into BON1 cells and luciferase
activity was measured with the Dual Luciferase Reporter
Assay System (Promega). The relative luciferase activity
was calculated by the ratio of luciferase/ β-gal activity. ELISA Total RNA was extracted from cells and tumor
tissues with Trizol reagent (Life Technology) according
to the manufacture’s instruction. 500 ng total RNA
was reverse transcribed using Oligo-dT under standard
conditions provided by the manufacture (Life technology). Quantitative real-time PCR was performed on a
LightCycler (Roche) using LightCycler FastStart DNA
Master SYBR Green Plus (Roche) in a final volume of
10 μl. Primer sequences and conditions for RT-PCR are
listed in Supplementary Table 1. Expression levels of
the housekeeping genes human β-actin were used for
normalization. Measurement of VEGF-A and IL-8 secretion in cell
culture supernatant was performed with ELISA kits (R&D
Systems, Wiesbaden, Germany) for human VEGF-A and
human IL-8 according to the manufacturer’s instruction. All the experiments were carried out in quadruplicates. Ubiquitination assay in cells COS7 cells were transfected with various plasmids as
indicated in individual experiment. 48 h after transfection,
cells were treated with 10 μM MG132 for 6 h, and the
whole cell lysates were prepared with immunoprecipitation
lysis buffer containing protease inhibitor cocktail and were
subjected to Immunoprecipitation with anti-HA antibody. The immunoprecipitated HA-PTEN were released from
the beads by boiling in SDS-PAGE sample buffer. The
analysis of PTEN ubiquitination was carried out by
immuno-blotting with anti-PTEN antibody. Tumor implantation manufacturer’s instructions. GST-PTEN expressed in
E.Coli DH5α and purified with glutathione-sepharose 4B
beads (GE Healthcare) as specified by the manufacture. The protein concentration was measured by Bradford
(Biorad) and then recombinant proteins were validated by
western blot for the correct expression. Female nude mice (4–6 weeks) were used for
tumor transplantation. Animal care followed institutional
guidelines and experiments were approved by local
animal research authorities. Mice were anaesthetized by
intraperitoneal administration of Ketavel and Rompun as
following the standard protocol. For tumor induction, the
pancreas was exposed and 50 μl matrigel and BON1 cell
mixture (1:1, 5 × 105) were injected into the head of the
pancreas. After 9 weeks, mice were sacrificed; primary
tumors and liver were collected. In vitro sumoylation conjunction assay was
performed using SUMO kit from Enzo bioscience. Specifically, 250 ng GST-PTEN and mutants, 1 μl of
SUMO2, 1 μl Aos1&Uba2, 1 μl Ubc9, and 2 μl 10×
reaction buffer with or without 1 μl ATP; H2O was added
to make the final volume of 20 μl. The reaction mixture
was incubated at 37°C for 2 hours and stopped by adding
sample loading buffer. The reaction mixture was separated
by SDS-PAGE and subsequently immunoblotted with anti-
PTEN antibody to detect SUMO modified PTEN. Sumoylation conjugation assay in cells COS7 cells were transfected with either control
plasmid or V5-RSUME plasmid together with His-
SUMO1/2 and V5-Ubc9. 48 hours post-transfection, cells
were harvested in PBS with protease inhibitor and divided by
two parts. 10% of the cells were preserved as input in SDS-
PAGE loading buffer and subjected to WB. The remaining
cells were first centrifuged then lysed in Ni-NTA lysis buffer
and subsequently subjected to protein purification by Nickel
magnetic Sepharose beads overnight at 4°C. The beads
were collected, washed 3 times with washing buffer and
the antigen-antibody complexes were recovered by boiling
in SDS-PAGE sample buffer. The input and samples were
subjected to Western blot with anti-PTEN antibody. Immunohistochemistry and immunofluorescence For immunocytofluorescence experiments, cells
or cryostat cut (5 μm) tumor tissues were fixed in 4%
paraformaldehyde for 5 minutes, and then blocked in
5% goat serum with 0.1% (v/v) triton X100 for 1 hour
at room temperature. Slides were incubated with the
indicated antibodies overnight at 4°C and then washed
and incubated with Alexa Fluor® 594 goat anti-rabbit
antibody (1:500, Invitrogen) or FITC-488 goat anti-mouse
(Invitrogen) antibody at room temperature for 2 hours. After washing with PBS, ProLong® Gold antifade reagent
with DAPI (Invitrogen) was used for mounting and
visualization of cell nuclei. Images were obtained using a
confocal microscope (Fluo View TM FV1000, Olympus). Images were obtained using 40× objectives. The
antibodies used for staining are listed in Supplementary
Table 4. For human pancreatic neuroendocrine tumors as
well as normal pancreas slides, paraffin embedded samples
were first subjected to deparaffinization and citric acid
based antigen retrieval was performed following standard
protocols. Sections were either stained with hematoxylin
and eosin (H&E) or subjected to immunohistochemistry
or immunofluorescence. Antibodies used in this study are
listed in Supplementary Table 4. Immunohistochemistry
images were obtained with light microscope (Zeiss,
Germany) with 20× objectives. Immunofluorescence
images were obtained using confocal microscope (Fluo
ViewTM FV1000, Olympus). Images were obtained using
60× objectives. In vitro sumoylation assay The His-tagged recombinant protein pQE30,
pQE30-RSUME were transformed into E. Coli M15
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Temporal Patterns of Voice Onset Time of English-
Sindhi Stops k Abbasi ( amabbasi@smiu.edu.pk )
ssatul Islam University Karachi https://orcid.org/0000-0002-0481-2992 Dr. Abdul Malik Abbasi ( amabbasi@smiu.edu.pk ) Temporal Patterns of Voice Onset Time of English-
Sindhi Stops
Dr. Abdul Malik Abbasi ( amabbasi@smiu.edu.pk )
Sindh Madressatul Islam University Karachi https://orcid.org/0000-0002-0481-2992
Dr. Mansoor Ahmed Channa
Quaid-e-Awaam University of Engineering, Science and Technology Nawab Shah
Dr. Imtiaz Husain
Sindh Madressatul Islam University Karachi
Ms Ahlam Khan
Ms Ahlam Khan
Research Article
Keywords: Voice onset time, voiceless, voiced, consonants, stops, place of articulation
Posted Date: August 22nd, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1744637/v2
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License Temporal Patterns of Voice Onset Time of English-
Sindhi Stops
Dr. Abdul Malik Abbasi ( amabbasi@smiu.edu.pk )
Sindh Madressatul Islam University Karachi https://orcid.org/0000-0002-0481-2992
Dr. Mansoor Ahmed Channa
Quaid-e-Awaam University of Engineering, Science and Technology Nawab Shah
Dr. Imtiaz Husain
Sindh Madressatul Islam University Karachi
Ms Ahlam Khan
Ms Ahlam Khan
Research Article
Keywords: Voice onset time, voiceless, voiced, consonants, stops, place of articulation
Posted Date: August 22nd, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1744637/v2
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Temporal Patterns of Voice Onset Time
of English & Sindhi Stops g
p
Abdul Malik Abbasi, Mansoor Ahmed Channa, Imtiaz Husain, Ahlam Khan Abstract ―The paper examines the temporal voice onset time (VOT) duration of consonantal stops
between the release burst of stops and the onset of vibration of the voice box. This is an acoustic
study of the Voice Onset Time of L1 Sindhi and L2 English stops. It aims to determine the acoustic
average values of Voice Onset Time (VOT) in Sindhi English stop consonants i.e., L1 and L2
production of speech. According to the author, this descriptive study on Voice Onset Time (VOT) in
Sindhi-English stop consonants has been conducted first time on English Sindhi VOT (ms) stops. The study recruited 10 (5 females and 5 male participants), who were Sindhi native speakers and
English as a Second Language (ESL) Learners. The stimuli were designed in which three voicing
pairs were selected from both Sindhi and English. The VOT (ms) of six Sindhi and English voicing
stop pairs /p/, /t/, /k/ & /b/, /d/, and /ɡ/ were measured i.e., one voiceless and one voiced. The
undergraduate students recorded their voice samples in both Sindhi and English. The data were
separately analyzed in Sindhi and English languages. There were acoustic differences within the
groups and between the groups in VOT (ms) of English and Sindhi. The statistical tests were run on
the results where the significance level of the p-value was fixed at <0.05. The results illustrate that
there is a significant difference between voiceless sounds i.e., /p/ & /k/ means. The data findings
reveal that there is no statistically significant difference between VOT (ms) means of males &
females for the voiceless & voiced consonantal sounds i.e., /t/, /b/, /d/ & /ɡ/. The data further
discovered that English Sindhi VOT characteristics of stops are not associated with gender; however,
English Sindhi VOT altered as a function of the place of articulation. Keywords: Voice onset time, voiceless, voiced, consonants, stops, place of articulation. Introduction This study aims to determine the voice onset time (VOT) average values of Indo-Aryan Speakers. It further
evaluates the effect of gender & place of articulation on VOT values. The main purpose of the study is to determine
and document the average acoustic values of VOT in Sindhi and English by Indo-Aryan Speakers with L1 Sindhi
mother tongue to add some new findings to the existing literature in the field. The voice onset time (VOT) study
documents the acoustic variations in temporal characteristics reflecting the physiological adjustment and articulatory
gestures. Voice Onset Time is well known acoustic characteristic to be the transmission between the release of an
oral compression which is also known as a noise burst and the onset of voicing. In acoustics when the stop
consonant is produced before voice onset, VOT (ms) is regarded to be positive VOT (ms), whereas, voicing onset
preceding the release is known to be negative VOT (ms). Voice onset time (VOT) is known to differ from the place
of articulation. The paper focuses on the time-based duration of three pair voicing stops between the release burst of
stops and the beginning of vibration of the voice box. It will also explore the variations in terms of the aspirated and
unaspirated stop consonants as reflected through voice onset timing (VOT); however, this reflection does not exist in
Pakistani English as the author is concerned. Since Pakistani English speakers or ESL learners do not aspirate stops
word-initially as English native speakers do. It will mainly be concerned with variations in VOT (ms) on account of
the place of articulation. The study focuses on the production of voicing onset time of English and Sindhi stops. The
study measured the voice onset time of English and Sindhi stop consonants. Both voice onset timings were analyzed
and compared to each other. According to the author, no study has been conducted on Voice Onset Time on Sindhi
stops nor VOT (ms) of L2 Pakistani English production. The studies have been carried out on VOT by different
researchers in the field for one language and almost not compared L1 speakers’ speech with L2. This paper
contributes the average values of VOT in terms of acoustic realizations of Sindhi-English VOT (ms) voicing pair
stops, which is a well-known phonological phenomenon in L1 and L2 production to the existing literature. Introduction Purpose The major purpose of the study is to document the acoustic durational average values of Voice Onset Time of L1
Sindhi and L2 English. Research Article License: This work is licensed under a Creative Commons Attribution 4.0 International License. R
d F ll Li License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Paper— Temporal Patterns of Voice Onset Time of English & Sindhi Stops g
Objectives of the Study •
To investigate voiceless stop sounds produced as pirated or unaspirated word-initially. g
p
p
p
p
y
xamine whether the VOT patterns produced by L1 and L2 speakers are associated the gender or not •
To examine whether the VOT patterns produced by L1 and L2 speakers are associated t •
To find out whether VOT is altered as a place of articulation or not. 1 Voice onset time (VOT) is demarcated as the break between the release of an oral contraction and the onset of
voicing. Once the stop is released before voice onset, the VOT (ms) is positive, whereas the voicing onset preceding
the release is regarded as a negative VOT (ms). English /p/, /t/, /k/ stop consonants are known as voiceless produced
as an aspirated stop at the beginning of a syllable without vocal cords voicing during the articulation. In contrast,
stop consonants /b/, /d/, /ɡ/ are produced voiced sounds vibrating the vocal cords. The VOT values of voiceless
plosive sounds in Turkish-speaking school children were positive and the VOT values of their voiced plosive sounds
were found negative. It was established that the height of the following vowel increases the VOT values of voiceless
plosive sounds and has a variable effect on voiced plosive sounds [1]. The average VOT (voice onset time) values of
the Turkish stop consonants by using 30 volunteers (15 female and 15 male) [2]. For this aim, the authors measured
the VOT values of six Turkish stops (i.e., /p/, /b/, /t/, /d/, /k/ and /ɡ/), which were uttered by 30 subjects in three
times, on wideband spectrograms. The average VOT values of /p/, /t/, /k/, /b/, /d/ and /ɡ/ were found to be 41, 66, 50,
53, 69, and 10 ms, respectively. The Voice Onset Time (VOT) is a time-based acoustic representative defined as the
time between the release of oral constriction for plosive production and the onset of vocal fold vibrations [3]. VOT
(ms) is also known to be the most reliable acoustic cue for the distinction between voiced and voiceless stops and
this temporal characteristic of stop consonants reflects the complex timing of supra-laryngeal coordination
[3]. ‘VOT (ms) is a measure of time at which periodic voicing begins relative to the time of the release of a
preceding stop or affricate and has proven to be an effective way of characterizing the laryngeal configuration of
these sounds’ [3]. g
Objectives of the Study Early research suggests that voiced and voiceless stops reveal longer negative and positive VOT
(ms) respectively before high, close vowels such as /i:/ and /u/. Voicing is relatively difficult to maintain in stops:
because air cannot escape from the oral cavity, the pressure differential across the larynx quickly falls below the
2cm/H2O required for voicing to occur. Both passive [4] and active [5] expansion of the vocal tract is often used to
help prolong voicing. VOT (ms) varies to some extent with a place of articulation. The further back the closure, the
longer the VOT (ms) [6]- [7]. The more extended the contact area, the longer the VOT (ms) [8]. The faster the
movement of the articulator, the shorter the VOT (ms) [9]. The voiced and voiceless terms can be misleading since
languages vary as to whether voicing is the distinguishable property or not all voiced or voiceless pairs involve a
voicing contrast least not in the phonetic sense. Languages have a two-way contrast in Obstruents, i.e., /p/ is distinct
from /p/, /v/ from /f/ etc. [10]. The traditional view of laryngeal contrast is known as Laryngeal Realism [9]- [11]. Furthermore, ‘The voicing group includes languages such as Japanese, Russian, and French. Then in the other
group, the group of so-called ‘aspiration’ languages, the contrast between sounds such as /b/ and /p/ does not strictly
speak, refers to the presence or absence of voicing. Instead, the distinguishable property is an aspiration, where /p/
can be aspirated (in strong syllables) whereas /b/ cannot, and importantly, both are phonetically voiceless. Therefore,
in these languages, the feature [voice] or the element |L| is not appropriate. To capture the relevant distinction
accurately, advocates of Laryngeal Realism prefer to use the feature [spread glottis] or the element |H| since these
refer to the presence of contrastive aspiration or voiceless-ness. The Languages in which aspiration occurs, include
the languages English, Swedish, and German’ [10]. Many phoneticians argue that vowels are usually longer before
voiced than before voiceless stops [12]- [13]- [14]- [15], and [16]. [17] and [16] suggest that one of the factors
contributing to VOT (ms) differences is the relative size of the supraglottal cavity behind the constriction point. That
includes the cavity behind the velar stop having a smaller volume than that behind the alveolar or bilabial stops. g
Objectives of the Study Secondly, the cavity in front of the velar stop has a larger volume than that in front of the alveolar or bilabial stops. Variations in VOT (ms) values are contact space between the articulators. The velar stops /k/ and /ɡ/ articulated
through the obstruction between the dorsum and the soft palate. In this regard, the contact space is prolonged as
compared to the bilabial and alveolar stops. Similarly, there is a phonological phenomenon in contact duration
between laminal vs. apical stops escorting dental vs. alveolar contrasts [18]. Iverson [19] contends that there are
some patterns like (1) the further back the closure, the longer the VOT (ms) [20]- [7], (2) the more extended the
contact area, the longer the VOT (ms) [21], and (3) the faster the movement of the articulator, the shorter the VOT
(ms) [9]. These can be seen in [18] classic cross-linguistic study of VOT (ms) but these are not the details regarding
the place of articulation. Sindhi is a language which has a full system of aspirated and unaspirated plosives as
different phoneme i.e., voiceless, plosives /p/, /pʰ/, /t/ & /tʰ/, /k/, /kʰ/ and voiced plosives /b/, /bʰ/, /d/, /dʰ/, /ɡ/, /ɡʰ/. Voice onset time (VOT) is demarcated as the break between the release of an oral contraction and the onset of
voicing. Once the stop is released before voice onset, the VOT (ms) is positive, whereas the voicing onset preceding
the release is regarded as a negative VOT (ms). English /p/, /t/, /k/ stop consonants are known as voiceless produced
as an aspirated stop at the beginning of a syllable without vocal cords voicing during the articulation. In contrast,
stop consonants /b/, /d/, /ɡ/ are produced voiced sounds vibrating the vocal cords. The VOT values of voiceless
plosive sounds in Turkish-speaking school children were positive and the VOT values of their voiced plosive sounds
were found negative. It was established that the height of the following vowel increases the VOT values of voiceless
plosive sounds and has a variable effect on voiced plosive sounds [1]. The average VOT (voice onset time) values of
the Turkish stop consonants by using 30 volunteers (15 female and 15 male) [2]. For this aim, the authors measured
the VOT values of six Turkish stops (i.e., /p/, /b/, /t/, /d/, /k/ and /ɡ/), which were uttered by 30 subjects in three
times, on wideband spectrograms. Recording Procedure Recording Procedure g
Participating candidates were given a brief introduction to the recording procedure, and they were also given a list of
token words used in the study for familiarizing themselves with them. Six indexing cards were written in English
and six in Sindhi script separately. Total cards were twelve (12×10×3=360) a pack of twelve cards in Sindhi script
was given to the participating candidates for recording in English & Sindhi. Six cards were shuffled after each for
English were recorded first given to the candidates for recording their voice samples in one go for all six after
shuffling them. Since there were three repetitions for each token word. After each turn, there was a gap of one
minute and second, turn and third respectively. After completing six turns of English token words and then Sindhi
word tokens were recorded. Additionally, the participants were in a quiet and calm setting, with no background
noise, in a closed room far from the noise of city traffic. The microphone used faced them at around six inches. They
were instructed to maintain a normal speech rate and to read the token sounds in isolation using a neutral focus, as in
ordinary talking conditions. Subjects were recorded directly onto a laptop computer Acer Travel Mate Core i3
system using the Praat speech processing tool [22], and a high-quality microphone. The complete six turns of
English token words and then Sindhi word tokens were recorded in isolation to get a natural rate of speech. The
participants had no speech-impairment problems as it was self-reported by the participants and observed by the
author as an English language teacher. g
Objectives of the Study The average VOT values of /p/, /t/, /k/, /b/, /d/ and /ɡ/ were found to be 41, 66, 50, •
Hypothesis: If English or Sindhi voiceless stops are produced word-initially by Sindhi L2 spea •
Hypothesis: If English or Sindhi voiceless stops are produced word-initially by Sindhi L2 speakers, then
voiceless /p/, /t/, and /k/stop sounds are produced unaspirated. •
Hypothesis: If the patterns of male-female stops are compared, then patterns should be associated with gender. •
Hypothesis: If English-English VOT is altered, it should function as a place of articulation. •
Hypothesis: If the patterns of male-female stops are compared, then patterns should be associated with gender. H
th
i
If E
li h E
li h VOT i
lt
d it h
ld f
ti
l
f
ti
l ti •
Hypothesis: If the patterns of male-female stops are compared, then patterns should be associated with gender. •
Hypothesis: If English-English VOT is altered, it should function as a place of articulation. 2 Method & Sampling Participating undergraduate students were recruited from an institution. The students were 10 in number (5 were
male and 5 were female). They all were Sindhi native speakers learning English as a second language. Their ages
ranged from 18 to 25 years. They had been exposed to learning and speaking English for about 10 years during their
academics at school, college, and the university. The participating level of English proficiency was the same as
determined by the author who is their English language teacher. Speech Stimuli p
Six English stops (/p/, /t/, /k/, /b/, /d/ and /ɡ/) English token words in voicing pairs along with English one vowel /ɔ/
and diphthong /əʊ/. Whereas six Sindhi stops /p/, /t/, /k/, /b/, /d/ and /ɡ/ were also selected in voicing pairs along
with total five Sindhi-English vowels /i:/, /o/, /ɒ/, /əʊ/, /ɔ:/. The token words were recorded in an isolation with three
repetitions. These voicing stop pairs were word-initial consonants. The stimuli were controlled by keeping all stop
sounds word-initially. Table 1. English Stops in Voicing Pairs Table 1. English Stops in Voicing Pairs SN
English
Voiceless Stops
English Words in
IPA
English Voiced
Stops
English Stops
IPA English
1
/p/
Poll
/pəʊl/
/b/
Bowl
/bəʊl/
2
/t/
Toll
/təʊl/
/d/
Dole
/dəʊl/
3
/k/
Call
/kɔ:l/
/ɡ/
Gall
/ɡ ɔ:l/
Table 2. Sindhi Stops in Voicing Pairs
SN
Sindhi Voiceless
Stops
Sindhi Words in IPA
Sindhi Voiced
Stops
Sindhi Words in IPA
1
/p/
/pi:rʊ/
/b/
/bi:rʊ/
2
/t/
/to:lʊ/
/d/
/do:lʊ/
3
/k/
/kɒ:no/
/ɡ/
/ɡɒ:no/ Results The results reveal that all the participants had a positive VOT (ms) when producing the voiced stop in Sindhi and
English. However, the results further reveal that Sindhi has voiceless unaspirated stops /p, t, k/ and ESL production
of stops word-initially. Sindhi native speakers do not produce English stop sounds aspirated words initially-medially
or at the coda position. This is on account of the reasons that there are different phonemes /pʰ/ & /p/ aspirated and
unaspirated stops in Sindhi. Therefore, the English stop sounds are also produced unaspirated by the native speakers
of Sindhi. The influence of gender, place of articulation and the identity of the following vowel on VOT (ms) mean
values were examined. The Sindhi bilabial sounds have 32 ms values, while alveolar sounds have 36 ms values and
velar values are 42. Whereas English sounds values are 36 for bilabial, 35 for alveolar, and 39 for velar sounds. Sindhi velar sounds are longer but not significantly different. On the contrary, English bilabial is longer than Sindhi
bilabial sounds but has no significant difference. These voicing stop pairs were word-initial consonants. Figure 1. illustrates the VOT (ms) values between Sindhi and English Sounds of male speakers. Both languages' VOT (ms)
values were analyzed as positive as follows: 3 3 Figure 1. Voiceless Sindhi & English stops: (/p/, /t/, /k/)
across male speakers
Fig 2. Voiceless Sindhi & English stops: (/b/, /d/, /ɡ/) across
male speakers
Figure 2 illustrates the VOT (ms) values between Sindhi and English Sounds. Both languages' VOT (ms) values are
analyzed as negative as follows: The Sindhi bilabial sounds have -94 ms values, while alveolar sounds have -89 ms
values, and velar values are -109. Whereas English sounds values are -95 for bilabial, -96 for alveolar, and -95 for
velar sounds. Figure 3 illustrates the VOT (ms) values between Sindhi and English Sounds of female speakers. Both
languages' VOT (ms) values are analyzed as positive as follows: The Sindhi bilabial sounds have 29 ms values,
while alveolar sounds have 29 ms values, and velar values are 37. Whereas English sounds values are 29 for
bilabial, 37 for alveolar, and 36 for velar sounds. 32
36
36
35
42
39
0
10
20
30
40
50
Sindhi
English
VOT (ms)
Bilabial
Alveolar
Velar
-94
-95
-89
-96
-109
-95
0
20
40
60
80
100
120
Sindhi
English
VOT (ms)
Bilabial
Alveolar
Velar Fig 2. Results Voiceless Sindhi & English stops: (/b/, /d/, /ɡ/) across
male speakers
-94
-95
-89
-96
-109
-95
0
20
40
60
80
100
120
Sindhi
English
VOT (ms)
Bilabial
Alveolar
Velar Figure 1. Voiceless Sindhi & English stops: (/p/, /t/, /k/)
across male speakers
32
36
36
35
42
39
0
10
20
30
40
50
Sindhi
English
VOT (ms)
Bilabial
Alveolar
Velar Fig 2. Voiceless Sindhi & English stops: (/b/, /d/, /ɡ/) across
male speakers Figure 1. Voiceless Sindhi & English stops: (/p/, /t/, /k/)
across male speakers Figure 2 illustrates the VOT (ms) values between Sindhi and English Sounds. Both languages' VOT (ms) values are
analyzed as negative as follows: The Sindhi bilabial sounds have -94 ms values, while alveolar sounds have -89 ms
values, and velar values are -109. Whereas English sounds values are -95 for bilabial, -96 for alveolar, and -95 for
velar sounds. Figure 3 illustrates the VOT (ms) values between Sindhi and English Sounds of female speakers. Both
languages' VOT (ms) values are analyzed as positive as follows: The Sindhi bilabial sounds have 29 ms values,
while alveolar sounds have 29 ms values, and velar values are 37. Whereas English sounds values are 29 for
bilabial, 37 for alveolar, and 36 for velar sounds. Figure 2 illustrates the VOT (ms) values between Sindhi and English Sounds. Both languages' VOT (ms) values are
analyzed as negative as follows: The Sindhi bilabial sounds have -94 ms values, while alveolar sounds have -89 ms
values, and velar values are -109. Whereas English sounds values are -95 for bilabial, -96 for alveolar, and -95 for
velar sounds. Figure 3 illustrates the VOT (ms) values between Sindhi and English Sounds of female speakers. Both
languages' VOT (ms) values are analyzed as positive as follows: The Sindhi bilabial sounds have 29 ms values,
while alveolar sounds have 29 ms values, and velar values are 37. Whereas English sounds values are 29 for
bilabial, 37 for alveolar, and 36 for velar sounds. Figure 2Figure 3. Voiceless Sindhi & English stops: (/p/, /t/,
/k/) across female speakers
Figure 3Figure 4. Voiced Sindhi & English stops: (/b/, /d/, /ɡ/)
across female speakers
Figure 4 illustrates the VOT (ms) values between Sindhi and English Sounds of female speakers. Results Both languages'
VOT (ms) values are analyzed as negative as follows: The Sindhi bilabial sounds have -94 ms values, while alveolar
sounds have-88 ms values, and velar values are -99. Whereas English sounds values are -104 for bilabial, -101 for
alveolar, and -102 for velar sounds. Statistical Tests Table 3 and Table 4 illustrate statistical values of VOT and
their differences between English and Sindhi speakers as follows:
29
29
29
37
37
36
0
10
20
30
40
Sindhi
English
VOT (ms)
Bilabial
Alveolar
Velar
-95
-104
-88
-101
-99
-102
80
85
90
95
100
105
110
Sindhi
English
VOT (ms)
Bilabial
Alveolar
Velar Figure 2Figure 3. Voiceless Sindhi & English stops: (/p/, /t/,
/k/) across female speakers
29
29
29
37
37
36
0
10
20
30
40
Sindhi
English
VOT (ms)
Bilabial
Alveolar
Velar Figure 3Figure 4. Voiced Sindhi & English stops: (/b/, /d/, /ɡ/)
across female speakers
-95
-104
-88
-101
-99
-102
80
85
90
95
100
105
110
Sindhi
English
VOT (ms)
Bilabial
Alveolar
Velar Figure 4 illustrates the VOT (ms) values between Sindhi and English Sounds of female speakers. Both languages'
VOT (ms) values are analyzed as negative as follows: The Sindhi bilabial sounds have -94 ms values, while alveolar
sounds have-88 ms values, and velar values are -99. Whereas English sounds values are -104 for bilabial, -101 for
alveolar, and -102 for velar sounds. Statistical Tests Table 3 and Table 4 illustrate statistical values of VOT and
their differences between English and Sindhi speakers as follows: Table 3 T-test on the differences between VOT (ms) values
(Male-Female)
Group Statistics
Gender
N
Mean
Std. Deviation
Std. Error
Mean
p
Male
10
33.70
3.164
1.001
Female
10
29.00
3.055
.966
t
Male
10
35.70
3.164
1.001
Female
10
33.20
5.959
1.884
k
Male
10
40.70
4.347
1.375
Female
10
36.30
3.268
1.033
b
Male
10
94.30
3.020
.955
Table 4 T-test on the differences between VOT (ms) values
(English Sindhi Speakers)
Group Statistics
Language
N
Mean
Std. Deviation
Std. Error
Mean
p
English
10
32.30
5.034
1.592
Sindhi
10
30.40
2.066
.653
t
English
10
36.20
5.007
1.583
Sindhi
10
32.70
4.138
1.309
k
English
10
37.50
3.598
1.138
Sindhi
10
39.50
5.017
1.586
b
English
10
99.20
7.285
2.304 Table 3 T-test on the differences between VOT (ms) values
(Male-Female)
Group Statistics
Gender
N
Mean
Std. Deviation
Std. Results Error
Mean
p
Male
10
33.70
3.164
1.001
Female
10
29.00
3.055
.966
t
Male
10
35.70
3.164
1.001
Female
10
33.20
5.959
1.884
k
Male
10
40.70
4.347
1.375
Female
10
36.30
3.268
1.033
b
Male
10
94.30
3.020
.955 Table 4 T-test on the differences between VOT (ms) values
(English Sindhi Speakers)
Group Statistics
Language
N
Mean
Std. Deviation
Std. Error
Mean
p
English
10
32.30
5.034
1.592
Sindhi
10
30.40
2.066
.653
t
English
10
36.20
5.007
1.583
Sindhi
10
32.70
4.138
1.309
k
English
10
37.50
3.598
1.138
Sindhi
10
39.50
5.017
1.586
b
English
10
99.20
7.285
2.304 4 Female
10
99.20
9.496
3.003
d
Male
10
92.30
8.858
2.801
Female
10
94.40
7.367
2.330
ɡ
Male
10
102.00
16.337
5.166
Female
10
100.80
8.496
2.687
Sindhi
10
94.30
6.800
2.150
d
English
10
98.30
6.881
2.176
Sindhi
10
88.40
5.777
1.827
ɡ
English
10
98.60
15.679
4.958
Sindhi
10
104.20
8.753
2.768
A separate t-test for the differences between VOT average values of male and female three pair voicing stops /p/, /t/,
/k/, /b/, /d/, /ɡ/ was carried out (t=3.746, df=18, p < .01) for the voiceless stops while (t=-.657, df=18, p < .01) for
the voiced stops. Levene’s test for equality of variances was run on the VOT values of English and Sindhi speakers
for voicing stop pairs /p/, /t/, /k/, /b/, /d/, /ɡ/ (t=3.379, df=18, for /p/; t=1.172, df=18, for /t/; t=2.558, df=18, for /k/;
whereas for the voiced stops, t=-1.555, df=18 for /b/; t=-.576, df=18 for /d/; t=.206, df=18 for /g/ p < .01). There is a
significant difference between voiceless sounds i.e., p & k means. However, there is no statistically significant
difference between VOT (ms) means of male & female for the voiceless & voiced consonantal sounds i.e., /t/, /b/,
/d/ & /ɡ/. Levene’s test for equality of variances was run on the VOT values of English and Sindhi speakers for
voicing stop pairs /p/, /t/, /k/, /b/, /d/, /ɡ/ (t=1.104, df=18, for /p/; t=1.704, df=18, for /t/; t=-1.024, df=18, for /k/;
whereas for the voiced stops, t=1.555, df=18 for /b/; t=3.484, df=18 for /d/; t=-.986, df=18 for / ɡ / p < .01). Results A separate t test for the differences between VOT average values of male and female three pair voicing stops /p/, /t/,
/k/, /b/, /d/, /ɡ/ was carried out (t=3.746, df=18, p < .01) for the voiceless stops while (t=-.657, df=18, p < .01) for
the voiced stops. Levene’s test for equality of variances was run on the VOT values of English and Sindhi speakers
for voicing stop pairs /p/, /t/, /k/, /b/, /d/, /ɡ/ (t=3.379, df=18, for /p/; t=1.172, df=18, for /t/; t=2.558, df=18, for /k/;
whereas for the voiced stops, t=-1.555, df=18 for /b/; t=-.576, df=18 for /d/; t=.206, df=18 for /g/ p < .01). There is a
significant difference between voiceless sounds i.e., p & k means. However, there is no statistically significant
difference between VOT (ms) means of male & female for the voiceless & voiced consonantal sounds i.e., /t/, /b/,
/d/ & /ɡ/. Levene’s test for equality of variances was run on the VOT values of English and Sindhi speakers for
voicing stop pairs /p/, /t/, /k/, /b/, /d/, /ɡ/ (t=1.104, df=18, for /p/; t=1.704, df=18, for /t/; t=-1.024, df=18, for /k/;
whereas for the voiced stops, t=1.555, df=18 for /b/; t=3.484, df=18 for /d/; t=-.986, df=18 for / ɡ / p < .01). It can be
concluded that there is no statistically significant difference between VOT (ms) means of English & Sindhi speakers
to produce the voiceless and voiced consonantal sounds i.e., /p/, /t/, /k/, /b/ & /ɡ/ whereas, there is a significant
difference observed for the voiced consonant sound ‘d’ means only. y
Table 5 T-test on the differences between VOT (ms) values (Male-Female & Voice) Table 5 T-test on the differences between VOT (ms) values (Male-Female & Voice)
Group Statistics
Gender
N
Mean
Std. Deviation
Std. Error Mean
Average Voiceless
Male
10
36.7100
1.62580
.51412
Female
10
32.8400
2.83361
.89607
Average Voiced
Male
10
96.1900
7.32339
2.31586
Female
10
98.1500
5.95357
1.88268 A separate t-test for the differences between VOT average values of male and female three pair voicing stops /p/, /t/,
/k/, /b/, /d/, /ɡ/ was carried out (t=3.746, df=18, p < .01) for the voiceless stops while (t=-.657, df=18, p < .01) for
the voiced stops. It can be concluded that there is no statistically significant difference between male & female
voiceless means whereas, there is a significant difference between the two voiced means. Results It can be
concluded that there is no statistically significant difference between VOT (ms) means of English & Sindhi speakers
to produce the voiceless and voiced consonantal sounds i.e., /p/, /t/, /k/, /b/ & /ɡ/ whereas, there is a significant
difference observed for the voiced consonant sound ‘d’ means only. Female
10
99.20
9.496
3.003
d
Male
10
92.30
8.858
2.801
Female
10
94.40
7.367
2.330
ɡ
Male
10
102.00
16.337
5.166
Female
10
100.80
8.496
2.687
Sindhi
10
94.30
6.800
2.150
d
English
10
98.30
6.881
2.176
Sindhi
10
88.40
5.777
1.827
ɡ
English
10
98.60
15.679
4.958
Sindhi
10
104.20
8.753
2.768 A separate t-test for the differences between VOT average values of male and female three pair voicing stops /p/, /t/,
/k/, /b/, /d/, /ɡ/ was carried out (t=3.746, df=18, p < .01) for the voiceless stops while (t=-.657, df=18, p < .01) for
the voiced stops. Levene’s test for equality of variances was run on the VOT values of English and Sindhi speakers
for voicing stop pairs /p/, /t/, /k/, /b/, /d/, /ɡ/ (t=3.379, df=18, for /p/; t=1.172, df=18, for /t/; t=2.558, df=18, for /k/;
whereas for the voiced stops, t=-1.555, df=18 for /b/; t=-.576, df=18 for /d/; t=.206, df=18 for /g/ p < .01). There is a
significant difference between voiceless sounds i.e., p & k means. However, there is no statistically significant
difference between VOT (ms) means of male & female for the voiceless & voiced consonantal sounds i.e., /t/, /b/,
/d/ & /ɡ/. Levene’s test for equality of variances was run on the VOT values of English and Sindhi speakers for
voicing stop pairs /p/, /t/, /k/, /b/, /d/, /ɡ/ (t=1.104, df=18, for /p/; t=1.704, df=18, for /t/; t=-1.024, df=18, for /k/;
whereas for the voiced stops, t=1.555, df=18 for /b/; t=3.484, df=18 for /d/; t=-.986, df=18 for / ɡ / p < .01). It can be
concluded that there is no statistically significant difference between VOT (ms) means of English & Sindhi speakers
to produce the voiceless and voiced consonantal sounds i.e., /p/, /t/, /k/, /b/ & /ɡ/ whereas, there is a significant
difference observed for the voiced consonant sound ‘d’ means only. Results Speaker 1
34
35
47
-97
-99
-117
Speaker 2
33
37
45
-96
-82
-101
Speaker 3
30
35
45
-93
-89
-113
Speaker 4
32
33
34
-89
-81
-112
Speaker 5
30
40
40
-95
-93
-101
English Stops VOT (ms) Across Male Speakers
English Speakers
p
t
k
b
d
ɡ
Speaker 6
37
31
39
-93
-108
-108
Speaker 7
38
33
43
-100
-99
-112
Speaker 8
36
34
38
-92
-84
-82
Speaker 9
30
39
41
-95
-89
-65
Speaker 10
37
40
35
-93
-99
-109
Discussion
The study was mainly concerned with variations in VOT (ms) due to place of articulation, differences between
and L2 (mother tongues) gender differences and voiced vs voiceless stop sounds This study has provided Speaker 1
34
35
47
-97
-99
-117
Speaker 2
33
37
45
-96
-82
-101
Speaker 3
30
35
45
-93
-89
-113
Speaker 4
32
33
34
-89
-81
-112
Speaker 5
30
40
40
-95
-93
-101
English Stops VOT (ms) Across Male Speakers
English Speakers
p
t
k
b
d
ɡ
Speaker 6
37
31
39
-93
-108
-108
Speaker 7
38
33
43
-100
-99
-112
Speaker 8
36
34
38
-92
-84
-82
Speaker 9
30
39
41
-95
-89
-65
Speaker 10
37
40
35
-93
-99
-109
on Speaker 1
34
35
47
-97
-99
-117
Speaker 2
33
37
45
-96
-82
-101
Speaker 3
30
35
45
-93
-89
-113
Speaker 4
32
33
34
-89
-81
-112
Speaker 5
30
40
40
-95
-93
-101
English Stops VOT (ms) Across Male Speakers
English Speakers
p
t
k
b
d
ɡ
Speaker 6
37
31
39
-93
-108
-108
Speaker 7
38
33
43
-100
-99
-112
Speaker 8
36
34
38
-92
-84
-82
Speaker 9
30
39
41
-95
-89
-65
Speaker 10
37
40
35
-93
-99
-109 Results Table 6 Voice Onset Time values for /p/, /t/, /k/ & /b/, /d/, / ɡ /
in Sindhi and English across ten speakers Table 6 Voice Onset Time values for /p/, /t/, /k/ & /b/, /d/, / ɡ /
in Sindhi and English across ten speakers Table 6 Voice Onset Time values for /p/, /t/, /k/ & /b/, /d/, / ɡ /
in Sindhi and English across ten speakers
Sindhi Stops VOT (ms) Across Female Speakers
Sindhi Speakers
p
t
k
b
d
ɡ
Speaker 1
31
30
39
-108
-87
-105
Speaker 2
29
29
37
-92
-88
-98
Speaker 3
29
31
32
-86
-86
-108
Speaker 4
28
31
35
-86
-95
-100
Speaker 5
28
26
40
-101
-84
-87
English Stops VOT (ms) Across Female Speakers
English Speakers
p
t
k
b
d
ɡ
Speaker 6
35
42
40
-116
-101
-106
Speaker 7
27
27
32
-106
-100
-111
Speaker 8
23
42
33
-100
-101
-108
Speaker 9
30
35
39
-99
-103
-98
Speaker 10
30
39
35
-98
-99
-87
Sindhi Stops VOT (ms) Across Male Speakers
Sindhi Speakers
p
t
k
b
d
ɡ Sindhi Stops VOT (ms) Across Female Speaker 5 Speaker 1
34
35
47
-97
-99
-117
Speaker 2
33
37
45
-96
-82
-101
Speaker 3
30
35
45
-93
-89
-113
Speaker 4
32
33
34
-89
-81
-112
Speaker 5
30
40
40
-95
-93
-101
English Stops VOT (ms) Across Male Speakers
English Speakers
p
t
k
b
d
ɡ
Speaker 6
37
31
39
-93
-108
-108
Speaker 7
38
33
43
-100
-99
-112
Speaker 8
36
34
38
-92
-84
-82
Speaker 9
30
39
41
-95
-89
-65
Speaker 10
37
40
35
-93
-99
-109
Discussion
The study was mainly concerned with variations in VOT (ms) due to place of articulation, differences between L1
and L2 (mother tongues), gender differences, and voiced vs. voiceless stop sounds. This study has provided a
descriptive account of voice onset time (VOT) for Sindhi L1 and L2 English voiced and voiceless series of stops. The acoustic study focuses on the production of voicing onset time of English and Sindhi stops. Both voice onset
timings were analyzed and compared to each other. Discussion The study was mainly concerned with variations in VOT (ms) due to place of articulation, differences between L1
and L2 (mother tongues), gender differences, and voiced vs. voiceless stop sounds. This study has provided a
descriptive account of voice onset time (VOT) for Sindhi L1 and L2 English voiced and voiceless series of stops. The acoustic study focuses on the production of voicing onset time of English and Sindhi stops. Both voice onset
timings were analyzed and compared to each other. According to the author, no study has been conducted on Voice
Onset Time on Sindhi stops nor VOT (ms) of L2 Pakistani English production. This paper contributes to the existing
literature on acoustic realizations of Sindh-English VOT (ms) stops, which is a well-known phonological
phenomenon in L1 and L2 production. There is a significant difference between voiceless sounds i.e., /p/ & /k/
means. However, there is no statistically significant difference between VOT (ms) means of male & female for the
voiceless & voiced stops i.e., /t/, /b/, /d/ & /ɡ/. It can further be concluded that there is no statistically significant
difference between VOT (ms) means of English & Sindhi speakers to produce the voiceless and voiced consonantal
sounds i.e., /p/, /t/, /k/, /b/ & /ɡ/ whereas, there is a significant difference observed for the voiced consonant sound
/d/ means only. Results According to the author, no study has been conducted on Voice
Onset Time on Sindhi stops nor VOT (ms) of L2 Pakistani English production. This paper contributes to the existing
literature on acoustic realizations of Sindh-English VOT (ms) stops, which is a well-known phonological
phenomenon in L1 and L2 production. There is a significant difference between voiceless sounds i.e., /p/ & /k/
means. However, there is no statistically significant difference between VOT (ms) means of male & female for the
voiceless & voiced stops i.e., /t/, /b/, /d/ & /ɡ/. It can further be concluded that there is no statistically significant
difference between VOT (ms) means of English & Sindhi speakers to produce the voiceless and voiced consonantal
sounds i.e., /p/, /t/, /k/, /b/ & /ɡ/ whereas, there is a significant difference observed for the voiced consonant sound
/d/ means only. References [1] A. Caramazza, Y. Grace H., Z. Edgar B and C. Ettore, "The acquisition of a new phonological contrast: The
case of stop consonants in French‐English bilinguals."," The Journal of the Acoustical Society of America, pp. 421-428., 1973. [2] . K. Nasukawa, ""A unified approach to nasality and voicing." In A Unified Approach to Nasality and
Voicing.," De Gruyter Mouton, 2012. [3] P. Ladefoged and I. Maddieson, The Sounds of the World's Languages, Oxford: Blackwell, 1996. [4] J. R. Westbury, "Enlargement of the supraglottal cavity and its relation to stop consonant voicing."," The
Journal of the Acoustical Society of America vol. 73, no. 4, pp. 1322-1336, 1983. [5] F. Bell− Berti, "Control of pharyngeal cavity size for English voiced and voiceless stops."," The Journal of the
Acoustical Society of America, vol. 57, no. 2, pp. 456-461, 1975. [6] M. Chen, "Vowel length variation as a function of the voicing of the consonant environment."," Ponetica, vol. 22, no. 3, pp. 129-159., 1970. [7] F. Öğü, A. K. Mehmet, Z. E. Erkan and M. Raşit, ""Voice onset times for Turkish stop consonants."," Speech
Communication, Vols. 1094-1099., no. 9, pp. 1094-1099., 2006. [8] G. E. Peterson and L. Ilse, "Duration of syllable nuclei in English."," The Journal of the Acoustical Society of
America, vol. 32, no. 6, pp. 693-703, 1960. [9] M. Halle and S. Kenneth, "Mechanism of glottal vibration for vowels and consonants."," The Journal of the
Acoustical Society of America, vol. 41, no. 6, pp. 1613-1613, 1967. [10] I. Maddieson, "Phonetic Universals. In The handbook of phonetic sciences," in Phonetic Sc
Blackwell, 1977, pp. 619-639. [11] P. Honeybone, "Diachronic evidence in segmental phonology: the case of obstruent larynge
2005. [12] E. Fischer-Jørgensen, ""Acoustic analysis of stop consonants."," Le Maître Phonétique , vol. 32, pp. 42-59,
1954. [13] I. T. Batuk and K. K. Mavis Emel, "Evaluation of the voice onset time in Turkish-speaking schoolchildren.","
International Journal of Pediatric Otorhinolaryngology, vol. 137, pp. 110-243, 2020. [14] L. Lisker and S. A. Arthur, "A cross-language study of voicing in initial stops: Acoustical measurements.","
Word, vol. 20, no. 3, pp. 384-422, 1964. [15] L. Lisker, "On “explaining” vowel duration variation."," In winter meeting of the Linguistic Society of America,
vol. 28, p. 225, 1973. [16] I. Maddieson, " "Further studies on vowel length before aspirated consonants."," UCLA Working Papers in
phonetics, vol. 38, pp. 82-90, 1977. [17] M. Halle and S. Conclusion It can be concluded that there is no statistically significant difference between male & female voiceless means
whereas, there is a significant difference between the two voiced means. Furthermore, there is no aspiration aspect
on initial stop sounds, though across tokens all words occur word-initially. Sindhi ESL learners did not produce the
aspirated stops across voiced and voiceless. There is no statistical difference across all stops except just d-voiced
alveolar consonantal sound. In the present study, the results indicated that gender is not a factor associated with
VOT (ms) in voiceless or voiced sounds with mother tongue Sindhi as compared to English L2. Whether or not this
is the characteristic realization of voicing in Sindhi or the effect of language/transfer effects from English remains to
be seen. Differences in VOT values are also on account of locations of word environment i.e., initial, medial, and
final. Therefore, it is important to mention anatomical/physiological implications of placement but also to
contextualize VOT in stops can vary depending on locations within the sound/word (e.g., initial, medial, final). The
data reveals further that their L2 speech production is reserved by the phonetic boundaries imposed by their existing
L1 phonological categories. By interpreting the data in this way, non-native production is the result of interference
from L1 experience, since L2 sounds are processed concerning the phonological categories established for the L1. Hypothesis # 1: If English or Sindhi voiceless stops are produced word-initially by Sindhi L2 speakers, then
voiceless /p/, /t/, and /k/stop sounds are produced unaspirated. The first hypothesis has been accepted that English
Sindhi VOT patterns of the voiceless stops were examined as unaspirated word-initially. Hypothesis # 2: If the
patterns of male-female stops are compared, then patterns should be associated with gender. The second hypothesis
has not been accepted that English Sindhi VOT patterns of stops are not associated with gender. Hypothesis # 3: If
English-English VOT is altered, it should function as a place of articulation. The third hypothesis has also been
accepted that English Sindhi VOT was altered as a function of the place of articulation. Additionally, English Sindhi
VOT characteristics of the voiced stops were found as negative word-initially. Statements and Declarations The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper. 6 References References Kenneth, "Mechanism of glottal vibration for vowels and consonants."," The Journal of the
Acoustical Society of America, vol. 41, no. 6, pp. 1613-1613, 1967. [18] T. Cho and L. Peter, "Variation and universals in VOT: evidence from 18 languages."," Journal of phonetics,
vol. 27, no. 2, pp. 207-229., 1999. [19] G. K. Iverson and S. Joseph C, "Aspiration and laryngeal representation in Germanic."," Phonology, vol. 12,
no. 3, pp. 369-396, 1995. [20] T. Cho and J. Sun-Ah, "Domain-initial strengthening as enhancement of laryngeal features: Aerodynamic
evidence from Korean."," UCLA working papers in phonetics, pp. 57-70, 2000. [21] G. E. Peterson and L. Ilse, "Duration of syllable nuclei in English."," The Journal of the Acoustical Society of
America, vol. 32, no. 6, pp. 693-703, 1960. [22] P. Boersma and W. David, "Praat: Doing phonetics by computer [Computer program](Version 6.1. 24).",
Accessed: August 22 (2022): 2022. 7 7
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https://openalex.org/W4313428790
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https://journals.ubmg.ac.id/index.php/JHTS/article/download/198/175
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English
| null |
OVERVIEW OF HEMOGLOBIN LEVELS IN PATIENTS WITH DIABETES MELLITUS Renal Complications in TOTO KABILA Hospital, 2020
|
Journal of Health, Technology and Science
| 2,022
|
cc-by
| 4,750
|
Puput Tilome1), Torajasa Achamar2) and Yolan Dunggio3)
1,3) Bina Mandiri University Gorontalo
2)Bone Bolango Health Office
E-mail: puputtilome14@gmail.com Puput Tilome1), Torajasa Achamar2) and Yolan Dunggio3)
1,3) Bina Mandiri University Gorontalo
2)Bone Bolango Health Office
E-mail: puputtilome14@gmail.com ABSTRACT In 2018 the number of sufferersDiabetes mellitusin Bone Bolango
district ranks sixth in Gorontalo Province. Data from Toto Kabila Hospital
in 2020, as many as 24 patients. In some cases the disease that can cause
complications in patients with diabetes mellitus is kidney failure. The
decrease in hemoglobin levels in patients with kidney disorders is caused by
decreased levels of erythropoietin which stimulates the bone marrow to
produce red blood cells. The aim of the study was to describe the hemoglobin levels in patients
with diabetes mellitus with kidney complications. This research design using quantitative descriptive. Conducted at Toto
Kabila Hospital with a total sample of 24 patients with diabetes mellitus
with kidney complications. The sampling technique was total sampling. Using the Univariate data analysis technique then the results are presented
in tabular form. Based on the results of the study of 24 samples, it was found that the
results of normal hemoglobin levels were 9 samples with a percentage
(37.5%) while patients who had abnormal hemoglobin levels were 15
samples with a percentage (62.5%). Keywords: hemoglobin, diabetes mellitus, kidney complications INTRODUCTION medical
nutrition
therapy,
physical
exercise
or
physical
activity
and
pharmacological intervention [22]. Non-Communicable Diseases (PTM)
is one of the major public health problems
in Indonesia which until now has become
a concern in the world of health because it
is one of the causes of death. This is
indicated by non-communicable diseases
that are increasing globally in the world
and nationally have occupied the top ten
diseases that cause death and most cases,
including
Diabetes
mellitus
(DM). Although Diabetes Mellitus is a chronic
disease that does not cause immediate
death, it can be fatal if handled
inappropriately. The
management
of
Diabetes Mellitus is known by four main
pillars, namely counseling or education, The number of people with diabetes
mellitus
has
increased
every
year,
especially
in
developing
countries. Judging from various studies that show a
tendency to increase the incidence of
diabetes mellitus in various parts of the
world. In (2013) there were 1.5 million
deaths caused by Diabetes Mellitus. In
(2018) there were 422 million people
worldwide who suffered from Diabetes
Mellitus, the majority of whom lived in
low and middle income countries [25]. In 2018 the prevalence of diabetes
mellitus based on doctor's diagnosis in 1 Overview of Hemoglobin Levels in Patients with Diabetes Mellitus, Kidney
Complications at Toto Kabila Hospital, 2020 Overview of Hemoglobin Levels in Patients with Diabetes Mellitus, Kidney
Complications at Toto Kabila Hospital, 2020 diabetic
nephropathy. Diabetic
nephropathy is a condition in which the
kidneys have decreased function and there
is damage to the blood-filtering membrane
caused by high blood sugar levels. which
explains where diabetic nephropathy is
found in diabetes mellitus patients in
about 34-54% [7]. people aged ≥ 15 years shows that
Gorontalo province is included in the
category of regions that are included in
the top 10 with the highest number of
people suffering from Diabetes Mellitus in
Indonesia with a percentage of 2.4% in
2018 [16]. There
were
4,415
people
with
Diabetes Mellitus found in Gorontalo
Province in 2018. Among them are
Pohuwato Regency with 563 inhabitants,
Boalemo Regency with 186 inhabitants,
North Gorontalo Regency with 211
people, Gorontalo Regency with 1.781
inhabitants, Gorontalo City with 302
people, Regency of Bonebolango with
1,372 people. The highest number of
patients was in the Gorontalo Regency
area with 1,781 patients and the least was
in the North Gorontalo Regency area, as
many as 211 people (Gorontalo Provincial
Health Office, 2018) [4]. INTRODUCTION Damage to the structure and function
of the kidneys can be accompanied by a
decrease in GFR / Glomerular Filtration
Rate. This decrease in GFR is related to
the clinical picture that will be found in
patients, one of which is a decrease in
hemoglobin levels in the blood which can
be said to be anemia. Hemoglobin is a
parameter of anemia, the decrease in Hb
levels in patients with kidney disorders is
caused
by
decreased
levels
of
erythropoietin, a hormone produced by
healthy kidneys to produce red blood cells
and if the body lacks oxygen levels,
healthy kidneys will release erythropoetin
hormone which will stimulate the marrow. the spine to produce more red blood cells
[22]. The number of diabetes mellitus
sufferers with kidney complications at
SOLOK Hospital in February-April 2017
showed that 15 male patients (50%) and
15 female patients (50%) [22]. and from
the results of research conducted on 30
male and female patients as many as 10
patients with diabetes mellitus with renal
complications who had hemoglobin levels
in the range of 10.1-11.0 gr / dl and most
of the hemoglobin levels were below
normal. This is because when the kidneys
are diseased or damaged they don't make
enough erythropoetin. As a result, the
bone marrow makes fewer red blood cells,
leading
to
anemia
or
decreased
hemoglobin levels [22]. Diabetics can experience various
long-term complications if their diabetes
is not managed properly. High blood sugar
for a long time will cause damage to
various organs, namely damage to the
blood vessels of the eye can cause visual
disturbances due to damage to the retina
of the eye (diabetic retinopathy), kidney
dysfunction can lead to kidney failure so
that patients must undergo dialysis,
attacks heart disease and stroke which can
lead to paralysis resulting in death and
amputation of the leg resulting in
disability [11]. Diabetes mellitus is a group of
diseasesmetabolism
which
is
characterized by hyperglycemia caused by
disorders of insulin secretion, insulin
action or both. Diabetes mellitus can also
cause chronic complications in parts of
human organs such as kidneys, eyes,
nerves and blood vessels. One of the
chronic microvascular complications is Diabetes mellitus incidence begins
with insulin deficiency as the main cause. On the other hand the emergence of
Diabetes mellitus can come from a
relative lack of insulin caused by insulin
resistance (Insuline Resistance). INTRODUCTION This
situation is characterized by the inability
of the organs to use insulin, so that insulin 2 Journal of Health, Technology and Science (JHTS)
E-ISSN: 2746-167X, Vol. 2, No. 3, September 2021 Puput Tilome, et. al. pp. 1-9 pp. 1-9 affects GFR and also indicates that there
are fewer functioning nephrons resulting
in impaired production of erythropoetin
produced
by
peritubular
fibroblasts. Erythropoetin stimulates the bone marrow
to make red blood cells, so that if there is
a disturbance in its formation, hemoglobin
is not maximally formed and anemia
results [15]. cannot function optimally in regulating
glucose metabolism. As a result, blood
glucose levels increase (hyperglycemia)
[3]. The greatness of Diabetes Mellitus
will appear more powerful when Diabetes
Mellitus enters the stage of complications. Diabetes mellitus can attack almost all
systems of the human body, from the skin
to the heart. The forms of complications
can be, respectively in the system: Anemia is a condition where the
hemoglobin level in the blood is below
normal which can be caused by a lack of
nutrients
for
blood
formation. complications
occur
most
often
in
diabetes mellitus patients, especially when
accompanied by nephropathy or renal
disorders. Chronic anemia causes tissue
hypoxia which is the key to diabetes
causing organ damage. Recent reports
have shown that anemia is a risk factor for
progression of End Stage Renal Disease
(ESRD) in patients with chronic kidney
disease, with or without diabetes. The
incidence of anemia increases with
increasing stage of ND and chronic kidney
disease [9]. 1. Cardiovascular system: hypertension,
myocardial infarction, coronary
insufficiency. 2. Eyes: Diabetic retinopathy 3. Nerves: Diabetic Neropathy 4. Kidney: pyelonephritis,
Glumeruloscelrosis 5. Lungs: tuberculosis 6. Liver: Cirrhosis of the liver 7. Skin: Gangrene, ulcers, furuncles The kidneys are the excretory organs
in vertebrates that are pea-like, located
behind the abdominal cavity, each kidney
consisting of the renal artery and renal The decrease in hemoglobin levels is
due to a chronic hyperglycemia condition
that can cause a hypoxic environment in
the renal interstitium, this kidney disorder 3 3 Overview of Hemoglobin Levels in Patients with Diabetes Mellitus, Kidney
Complications at Toto Kabila Hospital, 2020 Overview of Hemoglobin Levels in Patients with Diabetes Mellitus, Kidney
Complications at Toto Kabila Hospital, 2020 firoblasts decreases so that erythropoietin
production decreases which is the main
cause of anemia. vein. The macroscopic and microscopic
structure of the kidney consists of the
kidney, nephrons, blood vessels and renal
nerves. Macroscopic consists of an
excretory channel that leads to the
bladder, renal parenchyma that surrounds
the excretory tract, namely the medulla
and the renal cortex [19]. Nephron consists of glomerulus and
tubule. As part of the urinary system, the
kidneys have the main function of
maintaining the composition and volume
of body fluids in order to remain constant
through their excretory function. The
kidneys also have a specific function,
namely removing the waste products of
the body's metabolism, removing toxins
(drugs, food additives, etc.), filtering
impurities (especially urea) from the
blood and removing them together with
water in the form of urine [19]. When the diagnosis of diabetes
mellitus is confirmed, the possibility of
decreased kidney function should be
checked, as well as when the patient has
undergone routine treatment. Monitoring
recommended by the American Diabetes
Association (ADA) is checking for the
presence
of
microalbuminuria
and
determining
serum
creatinine
and
creatinine clearance. Diabetic nephropathy
is
a
microvascular
complication
of
diabetes mellitus that occurs in small
blood vessels. Diabetic nephropathy is one
of the leading causes of renal failure and
the
highest
mortality
among
all
complications of diabetes mellitus 6
Impaired diabetic renal function or
diabetic nephropathy is assessed by a
decrease in glomerular filtration rate
which can result in fibrosis and cause
anemia [3]. The decrease in the working period of
the kidneys is influenced by chronic
kidney
factors. 7. Skin: Gangrene, ulcers, furuncles 7. Skin: Gangrene, ulcers, furuncles Complications can be acute, and some
are chronic, acute complications are
characterized by: infection (Karbunke,
Angren,
pyolonephritis). Ketoacidosis
occurs, followed by coma. Chronic
complications associated with damage to
the walls of blood vessels that cause
atherosclerosis typical of the small blood
vessels at the end of the organ called
microangipathy, manifested in the form of
retinopathy,
glomeruloscelerosis
and
neuropathy [3]. The main function of the kidneys is to
filter and cleanse toxins in the body. In
one day the kidneys filter 200 liters of
blood, remove toxins and two liters of
water. The kidneys are also useful for
regulating blood pressure, regulating the
formation of red blood cells through the
hormone erythropoitiy, and playing a role
in maintaining the density of the dulan. If
the kidneys are damaged, people become
pale due to lack of blood, high blood
pressure, swelling and other possibilities
due to toxins that have accumulated in the
body,
including
bone
loss. Kidney
disorders
are
termed
Nephropathy
(Nephropathy). Kidney damage due to
diabetes is called diabetic nephropathy
[19]. neuropathy [3]. Hemoglobin
is
a
widely
used
parameter to determine the prevalence of
anemia. Determination of anemia status
using
only
hemoglobin
levels
is
incomplete, so it is necessary to add
another examination. hemoglobin is an
oxygen-carrying compound in red blood
cells. Hemoglobin can be measured
chemically and the amount of Hb / 100 ml
blood can be used as an index of the
oxygen-carrying capacity of the blood [1]. The decrease in hemoglobin levels is
due to a chronic hyperglycemia condition
that can cause a hypoxic environment in
the renal interstitium, this kidney disorder Hemoglobin
is
a
widely
used
parameter to determine the prevalence of
anemia. Determination of anemia status
using
only
hemoglobin
levels
is
incomplete, so it is necessary to add
another examination. hemoglobin is an
oxygen-carrying compound in red blood
cells. Hemoglobin can be measured
chemically and the amount of Hb / 100 ml
blood can be used as an index of the
oxygen-carrying capacity of the blood [1]. 7. Skin: Gangrene, ulcers, furuncles 60%
of
LPG
has
experienced an increase in plasma urea
and creatinine levels but patients still do
not feel some complaints until LPG below
30% of patients begin to show some
symptoms such as anemia, increased
blood pressure, disorders of phosphorus
and potassium metabolism accompanied
by nausea and vomiting , patients can also
get infections of the gastrointestinal tract,
water balance disorders such as hypo and
hypervolemia and electrolyte balance
disorders between sodium and potassium. Then until LPG below 15% of patients
begin to experience more serious signs
and symptoms requiring renal replacement
therapy such as dialysis or kidney
transplantation in this condition the
patient is said to have reached the end
stage of renal failure [24]. Chronic hyperglycemia can lead to
nonenzymatic glycation of amino acids
and proteins. Initially, glucose will bind to
the amino residues non-enzymatically,
then rearrange them to achieve a more
stable but still reversible form called
amadori
products. If
this
process
continues,
it
will
form
irreversible
Advanced
Glycation
End-Products
(AGEs). AGEs
are
thought
to
be
intermediaries for several cellular cellular
activities such as expression of adhesion
molecules that play a role in the
withdrawal of mononuclear cells, as well
as in the occurrence of cell hypertrophy. This process will continue until the
mesangium expansion occurs and the
formation of nodules and fibrosisi. In the
condition of kidney fibrosis the number of Decreasing
tissue
oxygen
concentration can result in the kidneys
increasing the production and release of
erythropoetin into blood plasma, which
stimulates stem cells to differentiate into
proeritroblasts, further increasing the rate
of mitosis, increasing the release of
reticulocytes from the spinal cord, and
inducing the formation of hemoglobin. In
kidney failure there is a deficiency of 4 Journal of Health, Technology and Science (JHTS)
E-ISSN: 2746-167X, Vol. 2, No. 3, September 2021 Journal of Health, Technology and Science (JHTS)
E-ISSN: 2746-167X, Vol. 2, No. 3, September 2021 Puput Tilome, et. al. pp. 1-9 pp. 1-9 damage to cells in the kidney as seen from
a decrease in the glomerular filtration rate,
the cells involved. one of which functions
as the formation of erythropoetin which is
useful in the production of red blood cells. Therefore, the lower the glomerular
filtration rate or the lower the kidney
function, the lower the hemoglobin level
is due to the inhibited erythropoietin
production activity [12]. erythropoietin. the process of forming
hemoglobin is reduced. 7. Skin: Gangrene, ulcers, furuncles There are other
factors in chronic kidney disorders that
also contribute to anemia, namely chronic
and acute inflammatory conditions that
have a strong influence on anemia of
chronic kidney disorders, by inflammatory
cytokines that decrease erythropoietin
production and induce apoptosis in
Colony Forming Units-Erythroid Cells
(CFU- E). At the initial induction of
apoptosis
CFU-E
cells
stop
the
development process into red blood cells. Cytokine inflammatory agents have also
been found to induce the production of
hepcidin, a peptide produced in the liver,
which interferes with red blood cell
production, by decreasing the availability
of iron for erythroblasts. This can reduce
the production of red blood cells [24]. Anemia is a condition where the
hemoglobin level in the blood is below
normal which can be caused by a lack of
nutrients
for
blood
formation. complications occur most often in patients
with diabetes mellitus, especially when
accompanied by nephropathy or renal
disorders. Chronic anemia causes tissue
hypoxia which is a key cause of diabetes
causing organ damage [18]. Anemia is functionally defined as a
decrease in the mass of erythrocytes
(redcell mass) so that it is unable to fulfill
its function to carry sufficient oxygen to
peripheral tissues. Practically anemia is
indicated by a decrease in hemoglobin,
hematocrit or erythrocyte count. But the
most
commonly
used
levels
are
hemoglobin, then hematocrit. It should be
noted that there are certain circumstances
in which these three parameters are
inconsistent with the erythrocyte mass,
such as dehydration, acute bleeding and
pregnancy [12]. Chronic Kidney Disease(CKD) is an
irreversible condition in which kidney
function decreases over time. CKD
(chronic kidney disease) usually develops
slowly and progressively, sometimes over
years, with patients often not realizing that
their condition is severe. The condition of
adequate function is impaired, resulting in
a decrease in the production of new red
blood cells and eventually anemia [23]. Decrease in hemoglobin levels due to
chronic hyperglycemia can cause a
hypoxic
environment
in
the
renal
interstitium, this kidney disorder affects
GFR and also indicates that there are
fewer functioning nephrons resulting in
impaired production of erythropoetin
produced by peritubular fibroblast cells. Erythropoietin
stimulates
the
bone
marrow to make red blood cells, so that if
there is a disturbance in its formation,
hemoglobin is not maximally formed and
anemia results [12]. RESEARCH METHODS This research is a descriptive research
with a quantitative approach. Where
descriptive research with a quantitative
approach is this research aims to explain
the existing phenomena by using numbers
to
rely
on
individual
or
group
characteristics to determine the value of
the independent variable, either one or
more (independent) variables without
making comparisons, or connecting with
variables other. This type of research is a
descriptive approach to see an overview of Impaired kidney function in diabetes
mellitus is assessed by a decrease in the
glomerular filtration rate which can result
in fibrosis and cause anemia, prolonged
hyperglycemia, which increases the risk of 5 5 Overview of Hemoglobin Levels in Patients with Diabetes Mellitus, Kidney
Complications at Toto Kabila Hospital, 2020 Overview of Hemoglobin Levels in Patients with Diabetes Mellitus, Kidney
Complications at Toto Kabila Hospital, 2020 the results of hemoglobin examination in
patients with diabetes mellitus with
kidney complications at Toto Kabila
Hospital [17]. This study uses univariate data
analysis techniques where the researcher
will describe or describe each research
variable then the results of the data
analysis are presented in the form of a
table along with a narrative [6]. Design This study used a cross
sectional design (cross section). Cross
sectional is a study by studying the
correlation between risk factors and
effects, carried out in situations where the
researcher intends to collect data from a
sample
or
population
(Notoatmodjo,
2012). Cross sectional research is used to
see the picturethe results of hemoglobin
examination in patients with renal-
complicating diabetes mellitus[13]. RESEARCH RESULT Based on research that has been
conducted on the image of hemoglobin
levels in patients with diabetes mellitus
with kidney complications at Toto Kabila
Hospital on October 8 - 19 October 2020
with a sample size of 24 patients, the
following results were obtained: Variable is something that becomes
the object of research observation, often
referred to as a factor that plays a role in
the research or symptoms to be studied. The variable in this study is the
hemoglobin level in patients with diabetes
mellitus with kidney complications [17]. Table 1. Results
of
Examination
of
Hemoglobin Levels in Patients
with
Complicated
Diabetes
Mellitus
Kidney. Table 1. Results
of
Examination
of
Hemoglobin Levels in Patients
with
Complicated
Diabetes
Mellitus
Kidney. Hemoglobin
Test Results
Frequency
%
Normal
9
37.5
Abnormal
15
62.5
total
24
100
Source: Data processed (2020) The population in this study were 24
patients with diabetes mellitus with
kidney complications at Toto Kabila
Hospital,
Bonebolango
Regency,
Gorontalo Province. The sample used in
this study was the entire population of
people with diabetes mellitus with kidney
complications at Toto Kabila Hospital,
Bonebolango
Regency,
Gorontalo
Province. Based on Table 1. it can be seen that,
patients with diabetes mellitus with
kidney complications who have normal
hemoglobin levels are 9 samples (37.5%)
while
patients
who
have
abnormal
hemoglobin levels are 15 samples with a
percentage (62.5%) at Toto Hospital. Kabila Bonebolango district in 2020. The sampling technique used in this
study was total sampling. Total sampling
is a sampling technique where the number
of samples is the same as the population. The research instrument is a tool that
researchers use in collecting data so that
jobs are younger and the results are better
[20]. The
instrument
used
is
the
Hematology Analizer tool which is used
as a measuring tool to determine the
results of hemoglobin levels in people
with
diabetes
mellitus
with
kidney
complications. In
addition,
the
questionnaire is used to determine the
characteristics
and
factors
that
can
interfere with research[20]. DISCUSSION Impaired kidney function in diabetes
mellitus is assessed by a decrease in GFR
which can result in fibrosis which causes
anemia,
long-lasting
hyperglycemia,
which increases the risk of damage to
cells in the kidney as seen from a decrease
in the glomerular filtration rate, one of
which is involved. which functions as the
formation of erythropoetin which is useful
in the production of red blood cells. If the 6 Journal of Health, Technology and Science (JHTS)
E-ISSN: 2746-167X, Vol. 2, No. 3, September 2021 Puput Tilome, et. al. pp. 1-9 Puput Tilome, et. al. pp. 1-9 pp. 1-9 in the range of 10.1-11.0 gr / dl and most
of the hemoglobin levels were below
normal. This is because when the kidneys
are diseased or damaged they don't make
enough erythropoetin. As a result, the
bone marrow makes fewer red blood cells,
causing anemia or decreased hemoglobin
levels. This decrease in GFR is related to
the clinical picture that will be found in
patients, one of which is a decrease in
hemoglobin levels in the blood. Decreased
hemoglobin levels in patients with kidney
disorders are caused by decreased levels
of erythropoetin, a hormone produced by
healthy kidneys to produce red blood cells
and if the body lacks oxygen levels,
healthy kidneys will release the hormone
erythropoetin which will stimulate the
bone marrow to produce more cells. red
blood [22]. body lacks oxygen levels, healthy kidneys
will release the hormone erythropoietin
which will stimulate the bone marrow to
produce more red blood cells.Therefore
the lower the glomerular filtration rate or
the lower the kidney function, the lower
the hemoglobin level because of the
inhibited
erythropoietin
production
activity. [12]. This study was conducted to examine
hemoglobin in patients with diabetes
mellitus with kidney complications. The
aim was to determine the hemoglobin
value and what factors could affect
hemoglobin levels in people with diabetes
mellitus with kidney complications at Toto
Kabila Hospital using an automatic
method with a total sample size of 24
diabetes
mellitus
patients. kidney
complications. Why this research is
considered important because it is an
effort to prevent or overcome anemia in
diabetes mellitus patients with kidney
complications, so hemoglobin checks are
necessary. Hemoglobin is used as an
initial screening test for anemia. In this study, 9 patients with diabetes
mellitus with kidney complications had
normal hemoglobin levels. Overview of Hemoglobin Levels in Patients with Diabetes Mellitus, Kidney
Complications at Toto Kabila Hospital, 2020 Overview of Hemoglobin Levels in Patients with Diabetes Mellitus, Kidney
Complications at Toto Kabila Hospital, 2020 Shibayama, Shinichi Antoku, Mariko
Abe,
Mizuo
Mifune,
Michiko
Togane.2010. Mild
Anemia
Is
Frequent and Associate With Micro-
and Macroangiopathies in Patients
With Type 2 Diabetes Mellitus. Journal of Diabetes Investigation. 1:
273-78. Shibayama, Shinichi Antoku, Mariko
Abe,
Mizuo
Mifune,
Michiko
Togane.2010. Mild
Anemia
Is
Frequent and Associate With Micro-
and Macroangiopathies in Patients
With Type 2 Diabetes Mellitus. Journal of Diabetes Investigation. 1:
273-78. CONCLUSION Based on the research that has been
done, it can be concluded that from 24
samples of diabetes mellitus patients with
kidney complications in Toto Kabila
Regional Hospital who were subjected to
hemoglobin examination and the results
obtained
were
15
samples
with a
percentage (62.5%) having decreased
hemoglobin levels. While the normal ones
were 9 patients with a percentage (37.5%). [10] Ministry
of
Health
RI. 2015. Indonesia Health Profile Report. Jakarta: Ministry of Health RI. [11] Marsden PA. Treatment of anemia in
chronic
kidney
disease-strategies
based on evidence. N Engl J Med. 2017; 261 (21): 2089-90. DISCUSSION One of the
influencing factors is the possibility that
the patient has previously received blood
transfusions but whose history is not
included in full in the medical record or
the other possibility that the patient has
previously
received
recombinant
erythropoietin, and factors that affect
patients with low hemoglobin levels are
usually caused because the patient's
condition
is
indeed
in
a
different
condition. severe
due
to
previous
underlying pain. The etiology of this
kidney failure is due to hypertension and
diabetes
mellitus. Chronic
kidney
sufferers are usually also disturbed by
their food intake, for example because of
loss of appetite, nausea, vomiting and
gastrointestinal disorders so that if not
monitored, the hemoglobin level will
continue to decrease over time. Decreased
renal function (characterized by an
increased stage and decreased GFR), the
more severe anemia will be [12]. This study used a sample of 24
patients with diabetes mellitus with
kidney complications in Toto Kabila
Hospital,
Bonebolango
Regency,
Gorontalo province in October 2020. a
percentage of 62.5% experienced a
decrease in hemoglobin levels. While 9
samples with a percentage of 37.5% were
in normal circumstances. These results
indicate that most respondents have
hemoglobin levels below normal. These results are in accordance with
previous research, it was found that the
number of diabetes mellitus sufferers with
kidney complications in SOLOK Hospital
in February-April 2017 showed that 15
male patients (50%) and 15 female
patients (50%) [22] . And from the results
of research conducted on 30 male and
female patients as many as 10 patients
with
diabetes
mellitus
with
kidney
complications who had hemoglobin levels 7 REFERENCES [1] Airam,
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Malik
Central
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[25] World Health Organization(WHO). (2018). Diabetes. (https://www.who.int/health-
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people with diabetes mellitus with 9 9
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Gender differences in sensory sensitivity in ASD patients aged 2 to 15
|
Research Square (Research Square)
| 2,023
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cc-by
| 6,342
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Gender differences in sensory sensitivity in ASD
patients aged 2 to 15 Niloufar Mohtasham Amiri
Mehdi Alizadeh
(
Alizadeh.m@iums.ac.ir
) Research Article Keywords: autism spectrum disorder (ASD), gender differences, sensory sensitivity, child development,
sensory processing, short sensory profile-2(SSP-2) Posted Date: July 19th, 2023 Results In taste smell sensitivity and visual auditory sensitivity higher scores (indicating higher sensitivity) were
found in females compared to males. Although movement sensitivity in females at younger ages was
less than in males, at older ages it has been found that females are more sensitive to movement.ASD
females showed higher scores at tactile sensitivity than males at older ages, compared to younger ages
between 9 to 11 years. Conclusion Sex differences in sensory sensitivity were observed in ASD children aged 2 to 15, with females being
more sensitive to ST/VA than males. On the other hand in total score and movement sensitivity, females
got more scores at older ages. These findings reinforce the need to take a developmental approach to
understand sex differences which may have diagnostic, prognostic and treatment implications. Methods 190 youth with ASD,(age range 2–15 years,131 male and 59 female) were assessed in the Iran Autism
Association clinic. The proportions of sensitivity present in Tactile / Taste Smell / Movement/ Visual and
Auditory items and also the TOTAL SCORE were analyzed as a binomial outcome and compared between
females and males. Patients were divided into four age groups and sensitivity by age group plots also
were analyzed. Background Despite the high prevalence of sensory processing difficulties in children with autism spectrum disorder
(ASD), research with a focus on the sex differences in sensory processing is limited contributing to later
diagnosis, referral, and interventions. This study aimed to assess the gender differences in sensory
sensitivity in ASD children. DOI: https://doi.org/10.21203/rs.3.rs-3132801/v1 DOI: https://doi.org/10.21203/rs.3.rs-3132801/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/16 1.1.Sex-bias, sex differences, and under-recognition of
autistic females Generally, males have been reported to be diagnosed four to five times more frequently than females;
however, a systematic review and meta-analysis study suggests lower male-to-female ratios that are
closer to 3:1 as females who would meet the criteria for autism may be at disproportionate risk of not
receiving a clinical diagnosis. (5) Several possibilities can lead to the under-recognition of autistic
females. First of all, females may present with partly different behavioral characteristics (6,7,8)that make it more
complicated for the autism phenotype to be recognized, diagnosed, and supported in an accurate and
timely manner. (9)In addition to different behavioral characteristics, clinically diagnosed autistic females
often present with coexisting emotional, behavioral, or cognitive difficulties including low intellectual level
(10,11)and higher rates of co-occurring medical conditions, including epilepsy in comparison to autistic
males. (6)As researchers often screen their participants to maximize the signal-to-noise ratio, females are
more likely to be excluded from research due to the higher frequency of co-occurring conditions like
epilepsy. As a result, Autism research studies have been dominated by male participants. So the male-to-
female participant ratio in research is even more exaggerated compared to the general population
prevalence ratio and it could limit the statistical power to detect small to moderate effects .(12)The outcome of this condition would be a ‘male-based’ understanding of autism(13) 12)The outcome of this condition would be a ‘male-based’ understanding o Furthermore, although hiding ASD symptoms or using compensatory behaviors to mitigate social
challenges including reducing repetitive behavior, ‘stimming’ or responses to sensory over-stimulation has
been reported in both gender(14,15,16,17), it is more common in females than males due to lack of
impaired neural self-representation and mentalizing (18). Considering these differences and a higher probability of hiding symptoms in females, it is necessary to
conduct more investigations on the differences between females and males with ASD to reduce Sex-bias
and under-recognition and reach an early diagnosis. 1.2. Sensory Processing, Sensory Over -Responsivity (SOR),
Under-responsivity (SUR), and Sensory Seeking in ASD 1. Introduction Autism spectrum disorder (ASD) is a common neurodevelopment condition that is characterized by early-
onset and persistent difficulties in social communication, sensory processing, and interaction along with
repetitive or stereotyped behaviors. (1). The prevalence of ASDs was reported to be 2.64% (95% CI = 1.91–
3.37), with 1.89% (95% CI = 1.43–2.36) in the general-population sample from regular schools and 0.75%
(95% CI = 0.58–0.93) in the high-possibility group from special education schools and a disability registry. Page 2/16 Page 2/16 1.2. Sensory Processing, Sensory Over -Responsivity (SOR),
Under-responsivity (SUR), and Sensory Seeking in ASD Sensory processing refers to the way we sense, perceive, and respond to sensory stimuli present in the
environment. This process is frequently referred to as a cascade that can be affected at different levels
for instance, how incoming information is detected, how it is processed and integrated into the brain and,
finally, how the ensuing behavior or response is modulated(19,20,21,22,23)Sensory processing disorders
(SPD) are diagnosed when a patient presents with significant difficulties or abnormalities in detecting, Page 3/16 modulating, interpreting or responding to sensory inputs(22)There are several methods for categorizing
SPD. The most common is a scheme based on sensory modulation patterns: hypo-responsiveness, which
refers to delayed responses or unresponsiveness to sensory stimuli; hyper-responsiveness, which is an
exaggerated or even aversive reaction to sensory stimuli; and sensory seeking, which refers to an unusual
fascination with the craving of sensory stimulation, often repetitive in nature (24,25,26,27,22,28)It has
been reported that over 96% of children with autism suffer from Sensory Processing Disorder in one or
multiple domains. These sensory behavioral problems range from mild to severe as well as
communication and social deficits and can endure through adulthood. (29,30)Despite its prevalence (over
96%) and its impact on functioning in daily and social life, little has been said about sex differences in
sensory processing in ASD and it is determined as one of the areas of ASD symptomatology which has
suffered by lack of studies on sex differences which could further drive the male-biased knowledge base. While a recent meta-analysis by Ben-Sasson et al. on sensory symptoms in ASD, it is not reported ‘Gender’
as a significant moderator in sensory processing disorders(31), Kumazaki et al. (32)reported higher
scores in female children on “taste, smell and touch response,” compared to their male counterparts using
the childhood autism rating scale.(CARS; 33) In a study by Osório et al.(34) children aged 2 to 12 and 11 months have been studied in sensory
processing, social participation, and praxis by using SPM(35,36)and SPM-Preschool (SPM-P,37,38)
questionaries. The results of this study suggest that ASD females might be more likely to avoid or to be
distressed by some auditory stimuli, to retreat from noisy environments, or to be distracted by sounds
that others do not notice. Differences in the Balance and Motion subscale suggest that females may
have more difficulties with movement coordination and postural control. 1.2. Sensory Processing, Sensory Over -Responsivity (SOR),
Under-responsivity (SUR), and Sensory Seeking in ASD Furthermore, females may have
more difficulties in processing and responding to tactile stimuli, even though their results show only a
slight trend. (34) The results from these studies made our team eager to evaluate the sensory profiles of females and
males by using more sensory instruments including short sensory profiles. This study aims to study sex
differences in sensory sensitivity in ASD children. For this purpose, we short sensory profile-2 (DUNN-
2014)(39). The Sensory Profile 2 (Dunn, 2014) is a set of norm-referenced, parent and teacher questionnaires
designed to assess the sensory processing patterns of children from birth through 14 years, and 11
months. There are five different forms selected based on age: 1) Infant Sensory Profile 2 – Birth to 6
months; 2) Toddler Sensory Profile – 7 to 35 months; 3) Child Sensory Profile 2–3 to 15 years; 4) Short
Sensory Profile 2–3 to 15 years; and 5) School Companion Sensory Profile 2–3 to 15 years. (39)In this
study we use short sensory profile-2 to evaluate children aged 2 to 14 and 11 months in Iran Autism
Association. 2.1. Ethics Statement Informed written consent was obtained for all participant's legal guardians by procedures approved by
the Iran University Of Medical Science Ethics Committee. 2.2. Participants: A total of 190 children were included in the analyses. Participants were recruited via research registries at
the Iran Autism Association. Registry participants are referred through Autism Check-up Project,
association clinics, and advocacy organizations to provide services for individuals with ASD. To acquire a
sufficient sample of female children with ASD, families traveled from across the country to participate in
this study. Children were eligible to take part in the study if they met the following inclusion criteria:(1) diagnosed
with ASD by a certified professional (psychiatrist, clinical psychologist)(2) aged between 2 and 14 years
11 months;(3) without an intellectual disability, as indicated by having an IQ above 70. (5) no other
comorbidity like epilepsy and acute medical or genetic conditions(4) living in Iran. Under these criteria,
190 ASD participants (131 males, and 59 females )have been chosen to participate in this study. 2. Method Page 4/16 2.3. Measures: In this study, parents or legal guardians responded to caregiver-report questionnaires(SSP-2). The SSP-2
contains is a shortened form of Dunn’s Sensory Profile caregiver questionnaire which has 38 items
organized into seven sub-scales: Tactile Sensitivity (TAC; 7 items), Taste/Smell Sensitivity (TSM; 4 items),
Movement Sensitivity (MOV; 3 items), Under responsive/Seeks Sensation (USS; 7 items), Auditory
Filtering (AFL; 6 items), Low Energy/Weak (LEW; 6 items), and Visual/Auditory Sensitivity (VAS; 5 items). These subs-scales represent observable child behaviors that are scored on a 1–5 rating scale based on
their frequency (1 being “Always” and 5 being “Never”). The score on each sub-scale and Total score can
be used to classify children’s level of sensory abnormality (Typical, Probably Difference, or Definite
Difference) based on score percentiles from a large normative sample of children without disabilities(40). Burns et al. reported the SSP was the second most utilized measure (28.0% of studies) after the full-
length SP, after a systematic review of 93 studies assessing sensory processing in ASD.(41)The SSP has
been used as the sensory phenotyping measure of choice in large-scale ASD projects such as the Autism
Speak Autism Treatment Network(42)and the EU-AIMS Longitudinal European Autism Project(43). However the reliability of each sub-scale was assessed by calculating Cronbach’s alpha for the 117-child
sample (α = 0.82– 0.89) which shows a large correlation between the unit-weighted sub-scale scores in
the sample (mean reported r = 0.53, range 0.25–0.72; 44), Williams ZJ et al. reported Sub scale alpha and
omega total values, based on commonly-cited recommendations for internal consistency reliability(45),
all satisfactory (> 0.7) which were notably homogenous (> 0.9) in TSM and LEW (46). As the language of
this questionnaire is English, we used the Persian version of Sensory Profile 2 which is the translated
form of this questionnaire. Mirzakhani et al.(47) studied Internal Consistency and Item Analysis of the
Persian Version of the Child Sensory Profile 2 in Vulnerable populations. In this study, the Discrimination Page 5/16 Page 5/16 index was satisfactory for all the items of the Child Sensory Profile-2. The values of Cronbach’s alpha
ranged from 0.795–0.919 in typical children and 0.617–0.901 in autistic children, and 0.792–0.920 in
children with learning disabilities. Based on this study, the Persian version of the Child Sensory Profile 2 is
a valid (discrimination with vulnerable populations) and reliable (internal consistency) tool for assessing
sensory processing. 2.3. Measures: (47) Despite the high reliability and validity of Children Sensory Profile − 2 which
includes 86 items, our team decided to use Short Sensory Profile-2 which includes 38 items from Children
Sensory Profile-2.its reduced participant burden relative to lengthier surveys, widespread use in the ASD
literature and easier use by an assistant in counseling sessions were the reasons behind selecting this
questionnaire for our research project. 2.4. Statistical analyses: First, Cronbach’s alpha was calculated to examine the internal consistency of the Persian versions of
Short Sensory Profile 2. In the next step, by the idea we got from the Mahenderian et al.(48) study, as
dichotomizing reduces the variability of parental expectation of appropriate frequency of the behaviors,
we decided to transform SSP-2 item scores into dichotomous variables. This transformation could cause
an increase in our confidence that a particular sensitivity is present or absent(48).To accomplish this,
each sub-scales score was divided into the number of components of the item. Then scores less than
2.49 were converted to “0” (corresponding to the Absence of Sensitivity), and scores between 2.50 to 5
were converted to “1” (corresponding to the Presence of Sensitivity). All analyses were performed using SAS 9.4 (2002–2010 by SAS Institute Inc., Cary NC, USA ) and
Minitab. we compared females and males in each sub-scale based on Data Means. Because the concept
of p has no meaning here as we have to consider the values with the expected value, and we have no
meaning for age here. That's why the mean is the best criterion for evaluating differences. we reported the
result of 4 sub-scales (TAC, TSM, MOV, VAS) and also the SSP-2 Total Score. We also examined the
sensitivity across ages in males and females by plots that showed scores in each age group. To
accomplish this, we divided participants into 4 age groups due to having the same participants in each
age group. Age groups include 1) 2 to 4;2) 5 to 8 ;3) 9 to 11; 4)12 to 15. 3.1. Study sample: A total of 190 children from age 2 to 15 (131 (69%) male and 59 (31%) female ) were included in the
analyses. Sample information and demographics are reported in Table 1. The internal consistency
estimates for each sub-scale and questionnaire were reported in Table 2. The internal consistency of the
sub-scales that we analyzed was above 0.70 and for the SSP-2 questionnaire was above 0.855 which
shows acceptable reliability estimates. Page 6/16 Page 6/16 Table 1
Baseline characteristics example
Characteristics
Number of participants
190
Age
9.67 ± 0.52
Gender
Male
131 (69%)
Female
59 (31%) Table 1 Table 2
Short Sensory profile-2 Internal consistency estimates (Crohnbach's
alpha)
Number of Items
Crohnbach’s alpha
-SSP2
38
0.855
Tactile Sensitivity
7
0.718
Smell Taste Sensitivity
4
0.889
Movement Sensitivity
3
0.857
Avoiding And Seeking
7
0.601
Auditory Filtering
6
0.678
Energy
6
0.898
Visual Auditory Sensitivity
5
0.791 Table 2
Short Sensory profile-2 Internal consistency estimates (Crohnbach's
alpha)
Number of Items
Crohnbach’s alpha
-SSP2
38
0.855
Tactile Sensitivity
7
0.718
Smell Taste Sensitivity
4
0.889
Movement Sensitivity
3
0.857
Avoiding And Seeking
7
0.601
Auditory Filtering
6
0.678
Energy
6
0.898
Visual Auditory Sensitivity
5
0.791
Sex differences in TAC, TSM, MOV, VAS, and Total
e:
ensitivity:
t of analyzing females and males in Tactile Sensitivity are presented in Table 3. When
g female and male participants in Tactile Sensitivity, males had significantly higher scor Table 2
Short Sensory profile-2 Internal consistency estimates (Crohnbach's
alpha) Table 2 3.2. Sex differences in TAC, TSM, MOV, VAS, and Total
Score: 3.2. Sex differences in TAC, TSM, MOV, VAS, and Total
Score: Tactile Sensitivity: The result of analyzing females and males in Tactile Sensitivity are presented in Table 3. When
comparing female and male participants in Tactile Sensitivity, males had significantly higher scores than
females at younger ages. (age group 2–4)whereas females have been reported notably more sensitive to
tactile sensation in ages 9–11. On the other hand, the pattern of sensitivity to age group, illustrates a rise
in men’s tactile sensitivity in older ages (12–15), while females were less sensitive after age 11. Page 7/16 Page 7/16 Taste Smell Sensitivity: Taste Smell Sensitivity: Based on the results (Table 4), in all age groups, females are more sensitive to Taste and Smell in
comparison to males and there was a significant difference in the effect of age between 9 to 11 years
old. Average Total Score: Table 7 represents the result of analyzing females and males in Total items including seek and avoid,
energy, auditory filtering in addition to sensory sensitivity items. In total scores, males have been reported
to get higher scores at younger ages whereas females got higher scores than males after age 4. Movement Sensitivity : The result of analyzing females and males in Movement Sensitivity are presented in Table 5. Among both
genders with ASD, Older children obtained higher scores in motion sensitivity than younger children. In
younger age groups (2–8) males were reported to be more sensitive than females, whereas, in groups
older than 9 years, females were reported to be more sensitive than males. The differences between
males and females reach their peak at age 9–11 females are more motion sensitive approximately two
times more than males. Both sexes have been more sensitive across age, but the magnitude was greater
in females. Visual/Auditory Sensitivity: 3.1. Study sample: Furthermore, in both gender, from the age of 2–4 to 5–8, children are getting more sensitive to Taste
and Smell and it reaches to its peak at 5–8 years old. Then the sensitivity to taste and smell tends to
decrease when they were getting older( after age 9 years old to 15) Movement Sensitivity : Visual/Auditory Sensitivity: Table 6 represents the result of analyzing females and males in Motion Sensitivity. Although females
have been reported to be more sensitive in all age groups, the differences between females and males
were remarkably seen in younger ages. (age group 2–4). The pattern of sensitivity to the age group was
similar between both genders and it showed a peak at 5 to 8 years old and a decrease in Visual/Auditory
sensitivity after 8 years old. Average Total Score: 4. Discussion In this study, we found that there were differences between females and males in sensory sensitivity. Females have been reported as more sensitive to Smell/Taste and Visual/Auditory Sensitivity. In addition,
considering age's effect on sensory sensitivity, females are more sensitive to Motion and Tactile
Sensitivity at older ages. This finding highlights the critical importance of examining age effects when
exploring diagnostic group differences in behavioral, functional, and Sensory Processing Disorders (SPD)
across ASD. we also assessed the internal consistency of the Persian version of Short Sensory Profile 2
which showed it is highly reliable. Page 8/16 Several reasons were suggested for differences in sensory sensitivity. One of the reasons could be
differences in brain patterns in females and males. To investigate whether sex differences in the
behavioral phenotype of ASD are linked to normative sex differences in brain neuroanatomy, Supekar k
and Menon V. explored the relationship between the multivoxel brain morphometry patterns and symptom
severity in girls and boys with ASD. Their findings indicate that the brains of girls with ASD are structured
differently from those of boys which are linked to sex differences in behavioral impairments like lower
severity of repetitive/restricted behaviors in girls. By using MVPA analysis, they found that the GM in
several cortical and subcortical regions could differentiate girls and boys with ASD. GM volume in the left
motor cortex, left supplementary motor area (SMA), left lingual/fusiform gyrus, left angular gyrus, right
insula, bilateral cerebellum, and bilateral amygdala (height p < 0.001, FWE corrected, extent p < 0.01)
notably showed high accuracies (85–90%) for distinguishing girls from boys with ASD. This study
showed a classification accuracy of 88 percent for the left lingual/fusiform gyrus which is generally
responsible for the higher processing of visual information. (49) Later, Supekar k et al. studied one of the largest functional brain imaging cohorts of females and males
with ASD, by using a novel spatiotemporal deep neural network ( stDNN ) model. It has been proved that
brain features associated with motor, language, and visuospatial attentional systems reliably distinguish
females with ASD from males and are contributing to their clinical symptoms in distinct ways. (50) Author’s contributions: Author’s contributions: Dr.Alizadeh and Dr.Amiri conceptualized and designed the study, conducted and oversaw the review of
abstracts and papers, drafted the initial manuscript, and reviewed and revised the manuscript. Mr.Miri
and Dr.Tavakoli conducted background literature searches, wrote portions of the manuscript, reviewed
abstracts and papers, and reviewed and revised the manuscript. Dr. Shirdel did the data analyzing, wrote
portions of the manuscript and handled final submission processes .All authors approved the final
manuscript as submitted and agree to be accountable for all aspects of the work. 5. Limitations and Future Directions Ethical Approval: Ethical Approval: The authors also would like to thank IAS Board’s members for granting the Autism Check-Up Project
which helped our team to involve more participants and create an Autism Friendly Network that includes
hundreds of clinics around the country to provide therapy for sensory problems. Competing Interests: The authors have no conflicts of interest to disclose. The authors have no conflicts of interest to disclose. Funding: Page 9/16 Page 9/16 This work was funded by Iran Autism Association, Tehran.IAS is an NGO that provides supports, and
financial aid and accelerates research in autism. This work was funded by Iran Autism Association, Tehran.IAS is an NGO that provides supports, and
financial aid and accelerates research in autism. Availability of data and materials: Anonymized data and materials are available upon request via the corresponding author. All authors have read and approved the final manuscript. Our most sincere thanks go to all the caregivers,
participants, and professionals of the Iran Autism Association, who helped us distribute the survey. Ethical Approval: The authors also would like to thank IAS Board’s members for granting the Autism Check-Up Project
which helped our team to involve more participants and create an Autism Friendly Network that includes
hundreds of clinics around the country to provide therapy for sensory problems. Competing Interests: The authors have no conflicts of interest to disclose. Author’s contributions: Dr.Alizadeh and Dr.Amiri conceptualized and designed the study, conducted and oversaw the review of
abstracts and papers, drafted the initial manuscript, and reviewed and revised the manuscript. Mr.Miri
and Dr.Tavakoli conducted background literature searches, wrote portions of the manuscript, reviewed
abstracts and papers, and reviewed and revised the manuscript. Dr. Shirdel did the data analyzing, wrote
portions of the manuscript and handled final submission processes .All authors approved the final
manuscript as submitted and agree to be accountable for all aspects of the work. Declarations Ethical Approval: Funding: This work was funded by Iran Autism Association, Tehran.IAS is an NGO that provides supports, and
financial aid and accelerates research in autism. Availability of data and materials: Availability of data and materials: Anonymized data and materials are available upon request via the corresponding author. All authors have read and approved the final manuscript. Our most sincere thanks go to all the caregivers,
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Scholar] Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table3to7.docx Page 16/16
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Differential Sandwich Theorems with Generalised Derivative Operator
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World Academy of Science, Engineering and Technology
International Journal of Mathematical and Computational Sciences
Vol:2, No:2, 2008
Differential Sandwich Theorems with Generalised
Derivative Operator
International Science Index, Mathematical and Computational Sciences Vol:2, No:2, 2008 waset.org/Publication/13046
1 Maslina
Darus and 2 Khalifa Al-Shaqsi
Abstract—In this paper, a generalized derivatives operator n
λ,β f
introduced by the authors will be discussed. Some subordination and
superordination results involving this operator for certain normalized
analytic functions in the open unit disk will be investigated. Our
results extend corresponding previously known results.
U). Recently Miller and Mocanu [5] obtained conditions on
h, q and ψ for which the following implication holds:
Keywords—Analytic functions, Univalent functions, Derivative
operator, Differential subordination, Differential superordination.
We now state the following definition.
(z ∈ U).
Definition 1.1: [3] Let function f in A, then for n, λ ∈ N0
and β > 0, we define the following differential operator
I. I NTRODUCTION
Denote by U the unit disk of the complex plane:
Dnλ,β f (z) = z +
U = {z ∈ C : |z| < 1}.
∞
[1 + β(k − 1)]n C(k, λ)ak z k ,
(z ∈ U),
k=2
Let H(U) be the space of analytic function in U. Let
An = {f ∈ H(U), f (z) = z + an+1 z n+1 + · · ·,
for (z ∈ U) with A1 = A.
For a ∈ C and n ∈ N we let
H[a, n] = {f ∈ H(U), f (z) = a + an z n + an+1 z n+1 + · · ·, }
(z ∈ U).
If functions f and F are analytic in U, then we say that f is
subordinate to F , and write f ≺ F , if there exists a Schwarz
function w analytic in U with |w(z)| < 1 and w(0) = 0 such
that f (z) = F (w(z)) in U. Furthermore, if the function F (z)
is univalent in U, then f (z) ≺ F (z) (z ∈ U) ⇔ f (0) = F (0)
and f (U) ⊂ F (U).
A function f , analytic in U, is said to be convex if it is
univalent and f (U) is convex.
Let p, h ∈ H(U) and let ψ(r, s, t; z) : C3 × U → C. If p and
ψ(p(z), zp (z), z 2 p (z); z) are univalent and if p satisfies the
(second-order) differential superordination
h(z) ≺ ψ(p(z), zp (z), z 2 p (z); z),
h(z) ≺ ψ(p(z), zp (z), z 2 p (z); z), ⇒ q(z) ≺ p(z)
(z ∈ U)
(1)
then p is called a solution of the differential superordination
(1). (If f subordinate to F , then F is superordinate to f ).
An analytic function q is called a subordinant of the differential
superodination, or more simply a subordinant if q ≺ p for all
p satisfying (1). A univalent subordinant q that satisfies q ≺ q
for all subordinants q of (1) is said to be the best subordinant.
(Note that the best subordinant is unique up to a rotation of
1,2 School
of Mathematical Sciences, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, Bangi 43600 Selangor D. Ehsan, Malaysia,
email: 1 maslina@ukm.my email: 2 ommath@hotmail.com
International Scholarly and Scientific Research & Innovation 2(2) 2008
where C(λ, k) = k+λ−1
.
λ
Special cases of this operator includes the Ruscheweyh
derivative operator D0λ,1 f (z) ≡ Dλ [6], the Salagean
derivative operator Dn0,1 f (z) ≡ Dn [2], the generalized
Salagean derivative operator Dn0,β f (z)
≡
Dβn [1]
and the generalized Ruscheweyh derivative operator
D1λ,β f (z) ≡ Dλ,β [4].
For n, λ ∈ N0 and β > 0, we obtain the following inclusion
relations:
n
n
Dn+1
λ,β f (z) = (1 − β)Dλ,β f (z) + βz(Dλ,β f (z)) , (2)
and
z(Dnλ,β f (z)) = (1 + λ)Dnλ+1,β f (z) − λDnλ,β f (z)(3)
The main object of the present paper is to find sufficient
condition for certain normalized analytic functions f to satisfy
q1 (z) ≺
n+1
f (z)
Dλ,β
n f (z)
Dλ,β
≺ q2 (z),
where n, λ ∈ N0 , β > 0 and q1 , q2 are given univalent
functions in U. Also, we obtain the number of known results
as their special cases.
In order to prove the original results we use shall need
the following definition and lemmas. In this paper unless
otherwise mentioned α, δ are complex numbers.
Definition 1.2: [5] Denote by Q, the set of all functions f
that are analytic and injective on U − E(f ), where
E(f ) = ζ ∈ ∂U : lim f (z) = ∞
z→ζ
and are such that f (ζ) = 0 for ζ ∈ ∂U − E(f ).
149
scholar.waset.org/1307-6892/13046
World Academy of Science, Engineering and Technology
International Journal of Mathematical and Computational Sciences
Vol:2, No:2, 2008
Lemma 1.3: [7] Let q be univalent in the unit disk U and θ
and φ be analytic in a domain D containing q(U) with φ(w) =
0 when w ∈ q(U). Set
ψ(z) = zq (z)φ(q(z))
and
and q is the best dominant.
Proof. Define the function p(z) by
h(z) = θ(q(z)) + ψ(z).
(9)
n+1
Dnλ+2,β f (z)
δ[2 − β(2 + λ)] Dλ,β f (z)
+ δβ(λ + 2)(λ + 1) n
n
β
Dλ,β f (z)
Dλ,β f (z)
If p is analytic with p(0) = q(0), p(U) ⊆ D, and
(4)
Dn+1
Dnλ+1,β f (z)
1
λ,β f (z)
+
[α
+
δ(1
−
)]
−δβ(λ + 1)
Dnλ,β f (z)
β
Dnλ,β f (z)
then
2
2
p(z) ≺ q(z)
International Science Index, Mathematical and Computational Sciences Vol:2, No:2, 2008 waset.org/Publication/13046
(z ∈ U).
Dnλ,β f (z)
Then the function p(z) is analytic in U and p(0) = 1.
Therefore, by making use of (2), (3) and (4), we obtain
Suppose that
1) ψ(z)
univalent in U, and
is starlike
zh (z)
2) Re ψ(z) > 0 for z ∈ U.
θ(p(z)) + zp (z)φ(p(z)) ≺ θ(q(z)) + zq (z)φ(q(z)),
Dn+1
λ,β f (z)
p(z) =
δ(1 − β)[1 − β(λ + 1)]
β
= δzp (z) + (δ + α)(p(z))2
−
and q is the best dominant.
Lemma 1.4: [8] Let q be convex univalent in the unit disk
U and ϑ and ϕ be analytic in a domain D containing q(U).
Suppose
that
(q(z))
> 0 for z ∈ U and
1) Re ϑϕ(q(z))
2) ψ(z) = zq (z)ϕ(q(z)) is starlike univalent in U.
By setting θ(w) = δw2 and φ(w) = α, it can be easily
observed that θ(w) and φ(w) are analytic in C − {0} and that
φ(w) = 0. Hence the result now follows by an application if
Lemma 1.3.
ϑ(p(z)) + zp (z)ϕ(p(z)) is univalent in U and
(5)
then q(z) ≺ p(z) and q is best subordinant.
II. S UBORDINATION
RESULTS
Let
n+1
δ[2 − β(2 + λ)] Dλ,β f (z)
β
Dnλ,β f (z)
Dnλ+2,β f (z)
+δβ(λ + 2)(λ + 1) n
Dλ,β f (z)
n
Dλ+1,β f (z)
−δβ(λ + 1)2
Dnλ,β f (z)
2
Dn+1
1
λ,β f (z)
+[α + δ(1 − )]
β
Dnλ,β f (z)
−
δ(1 − β)[1 − β(λ + 1)]
β
(7)
If f ∈ A satisfies
Ψ(λ, δ, α; z) ≺ δzq (z) + (δ + α)(q(z))2
then
Dn+1
λ,β f (z)
Dnλ,β f (z)
1+Az
Corollary 2.2: Let q(z) = 1+Bz
(−1 ≤ B < A ≤ 1) in
Theorem 2.1, further assuming that (6) holds. If f ∈ A then,
Ψ(n, λ, β, δ, α; z) ≺ δ
Using Lemma 1.3, we first prove the following theorem.
Theorem 2.1: Let n, λ ∈ N0 , β > 0 and q(z) be convex
univalent in U with q(0) = 1. Further, assume that
zq (z)
2δq(z)
+1+
> 0 (z ∈ U).
(6)
Re
α
q (z)
Ψ(n, λ, β, δ, α; z) =
By using (10) in (8), we have
δzp (z) + (δ + α)(p(z))2 ≺ δzq (z) + (δ + α)(q(z))2 .
If p(z) ∈ H[q(0), 1] ∩ Q with p(U) ⊆ D, and
ϑ(q(z)) + zq (z)ϕ(q(z)) ≺ ϑ(p(z)) + zp (z)ϕ(p(z)),
≺ q(z)
International Scholarly and Scientific Research & Innovation 2(2) 2008
(10)
(8)
⇒
Dn+1
λ,β f (z)
Dn
λ,β f (z)
≺
1+Az
1+Bz ,
Also, let q(z) =
1+z
1−z ,
(A − B)z
1 + Az
+ (δ + α)
2
(1 + Bz)
1 + Bz
and
Dn+1
λ,β f (z)
Dn
λ,β f (z)
≺
1+z
1−z ,
By taking q(z) =
⇒
Dn+1
λ,β f (z)
Dn
λ,β f (z)
≺
1+z
1−z
is the best dominant.
2δz
1+z
+ (δ + α)
(1 − z)2
1−z
and
1+z
1−z
Ψ(n, λ, β, δ, α; z) ≺
,
then for f ∈ A we have,
Ψ(n, λ, β, δ, α; z) ≺
⇒
1+Az
1+Bz
2
1+z
1−z
μ
,
is the best dominant.
, (0 < μ ≤ 1), for f ∈ A, we have
2δμz
1+z
2
(1 − z) 1 − z
μ
2
, and
III. S UPERORDINATION
1+z
1−z
μ
μ−1
+ (α + δ)
1+z
1−z
is the best dominant.
AND SANDWICH RESULTS
Now, by applying Lemma 1.4, we prove the following
theorem.
Theorem 3.1: Let q be convex univalent in U with q(0) = 1.
Assume that
2(δ + α)q(z)q (z)
> 0.
(11)
Re
δ
Let f ∈ A,
150
Dn+1
λ,β f (z)
Dn
λ,β f (z)
∈ H[q(0), 1] ∩ Q.
scholar.waset.org/1307-6892/13046
2μ
,
World Academy of Science, Engineering and Technology
International Journal of Mathematical and Computational Sciences
Vol:2, No:2, 2008
Further, let Ψ(n, λ, β, δ, α; z) given by (7) be univalent in U
and
then
q1 (z) ≺
(δ + α)(q(z))2 + δzq (z) ≺ Ψ(n, λ, β, δ, α; z)
Dn+1
λ,β f (z)
Dnλ,β f (z)
,
For q1 (z) =
Proof. Theorem 3.1 follows by using the same technique to
prove Theorem 2.1 and by an application of Lemma 1.4.
International Science Index, Mathematical and Computational Sciences Vol:2, No:2, 2008 waset.org/Publication/13046
A and
satisfies. If
1+Az
1+Bz (−1
Dn+1
λ,β f (z)
Dn
λ,β f (z)
∈ H[q(0), 1] ∩ Q and
≺ Ψ2 (A2 , B2 , n, λ, β, δ, α; z)
then
2
Dn+1
1 + A2 z
1 + A1 z
λ,β f (z)
≺ n
≺
1 + B1 z
Dλ,β f (z)
1 + B2 z
δ(A − B)z
+
≺ Ψ(n, λ, β, δ, α; z)
(1 + Bz)2
where
1 + Az
≺
1 + Bz
1+Az
1+Bz ,
1 + A1 z
Ψ1 (A1 , B1 , n, λ, β, δ, α; z) := (δ + α)
1 + B1 z
Dn+1
λ,β f (z)
Dnλ,β f (z)
+
is the best subordinant.
Dn+1
λ,β f (z)
Dn
λ,β f (z)
1+z
1−z ,
f ∈ A and
Also, by let q(z) =
Q. Further assuming that (11) satisfies. If
2δz
1+z
+ (δ + α)
(1 − z)2
1−z
1+z
1−z
and
1+z
1−z
≺
Dn+1
λ,β f (z)
,
Dn
λ,β f (z)
Finally, by taking q(z) =
Dn+1
λ,β f (z)
Dn
f (z)
λ,β
and
satisfies. If
1+z
1−z
μ
2
∈ H[q(0), 1] ∩
+
≺ Ψ(n, λ, β, δ, α; z),
is the best subordinant.
1+z
1−z
μ
, (0 < μ ≤ 1), f ∈ A
δ(A1 − B1 )z
,
(1 + B1 z)2
1 + A2 z
Ψ2 (A2 , B2 , n, λ, β, δ, α; z) := (δ + α)
1 + B2 z
2
2
δ(A2 − B2 )z
.
(1 + B2 z)2
1+A2 z
1z
Hence 1+A
1+B1 z and 1+B2 z are respectively the best subordinant and best dominant.
Remark 3.5: For special cases of the above results follows
by choosing different values of n, λ and β.
∈ H[q(0), 1] ∩ Q. Further assuming that (11)
ACKNOWLEDGMENT
The work presented here was fully supported by eSciencefund: 04-01-02-SF0425, MOSTI, Malaysia.
2δμz
1+z
2
(1 − z) 1 − z
⇒
1+A2 z
1+B2 z ,
Ψ1 (A1 , B1 , n, λ, β, δ, α; z)
≺ Ψ(n, λ, β, δ, α; z)
≤ B < A ≤ 1), f ∈
then
⇒
q2 (z) =
Corollary 3.4: If f ∈ A,
∈ H[q(0), 1] ∩ Q. Further assuming that (11)
1 + Az
(δ + α)
1 + Bz
and
1+A1 z
1+B1 z ,
we have the following corollary.
By using Theorem 3.1, we have the following corollary.
Dn+1
λ,β f (z)
Dn
λ,β f (z)
≺ q2 (z),
where (−1 ≤ B2 ≤ B1 < A1 ≤ A2 ≤ 1),
and q is the best subordinant.
Corollary 3.2: Let q(z) =
n f (z)
Dλ,β
and q1 and q2 are respectively the best subordinant and best
dominant.
then
q(z) ≺
n+1
f (z)
Dλ,β
, and
μ−1
1+z
1−z
+ (α + δ)
μ
≺
1+z
1−z
Dn+1
λ,β f (z)
Dn
λ,β f (z)
2μ
≺ Ψ(n, λ, β, δ, α; z),
is the best subordinant.
Combining the results of differential subordination and superordination, we state the following Sandwich Theorems .
Theorem 3.3: Let q1 and q2 be convex univalent in U and
Dn+1 f (z)
∈
satisfies (11) and (6), respectively. If f ∈ A, Dλ,β
n f (z)
λ,β
H[q(0), 1] ∩ Q and Ψ(n, λ, β, δ, α; z) is univalent in U, and
δzq1 (z) + (δ + α)(q1 (z))2 ≺ Ψ(n, λ, β, δ, α; z)δzq2 (z)
(12)
+(δ + α)(q2 (z))2 ,
International Scholarly and Scientific Research & Innovation 2(2) 2008
R EFERENCES
[1] F. M. Al-Oboudi, On univalent functions defined by a generalized
Sălăgean operator, Ind. J. Math. Math. Sci. 27, (2004), 1429-1436.
[2] G. Ş. Sălăgean, Subclasses of univalent functions, Lecture Note in
Math.(Springer-Verlag), 1013, (1983), 362-372.
[3] K. Al-Shaqsi and M. Darus, Differential subordination with generalized
derivative operator. (Submitted)
[4] K. Al Shaqsi and M. Darus, On univalent functions with respect to
k-symmetric points defined by a generalized Ruscheweyh derivatives
operator.(Submitted)
[5] S. S. Miller and P. T. Mocanu, Subordinants of differential superordinations, Complex Variables Theory Appl. 48(10), (2003), 815-826.
[6] St. Ruscheweyh, New criteria for univalent functions, Proc. Amer. Math.
Soc. 49, (1975), 109-115.
[7] S. S. Miller and P. T. Mocanu, Differential Subordinations: Theory and
Applications, Marcel Dekker Inc., New York, 2000.
[8] T. Bulboacă, Classes of first order differential superordinations, Demonstratio Math. 35(2), (2002), 287-292.
151
scholar.waset.org/1307-6892/13046
World Academy of Science, Engineering and Technology
International Journal of Mathematical and Computational Sciences
Vol:2, No:2, 2008
Maslina Darus received her Bachelor degree in Mathematics from Acadia
University, Canada, in 1992 and PhD in Pure Mathematics (Complex
Analysis) from University of Wales, Swansea, U. Kingdom in 1996.
Currently, she is a Professor of Mathematics in School of Mathematical
Sciences, Faculty of Science and Technology, Universiti Kebangsaan
Malaysia. She is also holding a post as Head of Mathematics program. Her
special interest is in the geometric function theory and its applications.
International Science Index, Mathematical and Computational Sciences Vol:2, No:2, 2008 waset.org/Publication/13046
Khalifa Al-Shaqsi is currently a PhD student under supervision of Professor
Maslina Darus. He obtained Master degree in Mathematics at School of
Mathematical Sciences, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia in 2006.
International Scholarly and Scientific Research & Innovation 2(2) 2008
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https://openalex.org/W2154598603
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https://europepmc.org/articles/pmc4316799?pdf=render
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English
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De novo assembly of bacterial transcriptomes from RNA-seq data
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Genome biology
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cc-by
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Introduction genome sequence is available since reference-based ap-
proaches are fast and relatively precise. De novo assem-
bly involves assembling transcripts from sequencing
reads by combining overlapping reads. De novo assem-
bly is necessary when a high-quality reference genome
is unavailable, such as for many non-model organisms,
when analyzing complex microbial communities, in meta-
transcriptome studies, and when investigating uncultur-
able microorganisms. High-throughput RNA sequencing (RNA-seq) is being
used increasingly for transcriptome assays [1]. One of the
challenges for studies employing RNA-seq experiments is
efficient and reliable extraction of transcriptomic insights
from the wealth of RNA-seq data. Often, following RNA-
seq experiments, the large resulting data sets are subjected
to various stages of computational analysis, such as quality
control, normalization, transcriptome assembly, quantifi-
cation of transcript abundance, and testing for differential
gene expression under various conditions [2]. Analysis
of the data can be a bottleneck in RNA-seq studies
owing to the size of the data, the complexity of the ana-
lysis, and a lack of user-friendly software tools. A number of mature computational tools exist for
both reference-based transcriptome assembly [5-7] and
de novo transcriptome assembly [8-11]. However, most of
the aforementioned tools were designed primarily for
eukaryotic transcriptomes. Bacterial transcriptome assem-
bly faces different challenges than eukaryotic transcriptome
assembly. For example, bacterial genomes are generally
denser than eukaryotic genomes and neighboring bacterial
transcripts frequently overlap, making it challenging to
distinguish the boundaries of neighboring bacterial tran-
scripts. Polycistronic messages further complicate bacter-
ial transcriptome assembly, particularly when different
promoters of an operon are employed under different
conditions. Also, models for noncoding RNAs in eukary-
otes are generally inappropriate for the small regulatory
RNAs common in bacteria. In particular, assembling transcripts is often a core
stage of RNA-seq data analysis, yet efficient and accurate
transcriptome assembly remains a challenging problem
owing to a variety of factors, including artifacts from li-
brary construction, errors in sequencing, variable intra-
read and inter-read error rates, repeat sequences, and
transcript expression ranges that span several orders of
magnitude [3]. Most approaches for assembling tran-
scripts from short read sequences relate to one of two
families: reference-based assembly and de novo assembly
[4]. Reference-based assembly involves aligning sequen-
cing reads to a sequenced reference genome. SOFTWARE SOFTWARE Open Access De novo assembly of bacterial transcriptomes
from RNA-seq data Brian Tjaden Abstract Transcriptome assays are increasingly being performed by high-throughput RNA sequencing (RNA-seq). For
organisms whose genomes have not been sequenced and annotated, transcriptomes must be assembled
de novo from the RNA-seq data. Here, we present novel algorithms, specific to bacterial gene structures and
transcriptomes, for analysis of bacterial RNA-seq data and de novo transcriptome assembly. The algorithms are
implemented in an open source software system called Rockhopper 2. We find that Rockhopper 2 outperforms
other de novo transcriptome assemblers and offers accurate and efficient analysis of bacterial RNA-seq data. Rockhopper 2 is available at http://cs.wellesley.edu/~btjaden/Rockhopper. Correspondence: btjaden@wellesley.edu
Computer Science Department, Wellesley College, Wellesley, MA 02481, USA © 2015 Tjaden; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Tjaden Genome Biology (2015) 16:1
DOI 10.1186/s13059-014-0572-2 Tjaden Genome Biology (2015) 16:1
DOI 10.1186/s13059-014-0572-2 p
g
y
p
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Introduction Reference-
based assembly is generally preferable when a high-quality In an attempt to address the paucity of computational
methods for assembling bacterial transcriptomes from
RNA-seq data, we previously developed Rockhopper
[12], a system that supports reference-based assembly of
bacterial transcriptomes. In the current study, we have Correspondence: btjaden@wellesley.edu
Computer Science Department, Wellesley College, Wellesley, MA 02481, USA Page 2 of 10 Tjaden Genome Biology (2015) 16:1 developed novel algorithms for de novo assembly of bac-
terial transcriptomes, which we have implemented in the
system Rockhopper 2. We show that our algorithms for
de novo assembly of bacterial transcriptomes outperform
other leading approaches, in terms of both sensitivity
and specificity. Further, our algorithms offer dramatic
improvements in efficiency, so that our de novo assem-
bly is comparable to reference-based assembly in terms
of execution time. While many de novo assemblers require
high-performance computing platforms, Rockhopper 2
has been designed with limited resource requirements so
that it performs effectively on common laptop machines. In addition to de novo transcriptome assembly, Rockhopper
2 is a comprehensive system that supports the various
stages of RNA-seq data analysis, including normalizing
data from different experiments, quantifying transcript
abundance, and testing for differential transcript expres-
sion. Details of the Rockhopper 2 workflow are illustrated
in Figure 1. Finally, we developed Rockhopper 2 with
user-friendliness in mind, so that it would be accessible to
a broad range of scientists that use bacterial RNA-seq ex-
periments in their investigations. Rockhopper 2 is open-
source software implemented in Java, released under the
GNU GPL license, and is available for all major platforms
at [13,14]. integration of the two structures, working in concert,
that enables Rockhopper 2’s speed and minimal mem-
ory usage, distinguishing it from other de novo assem-
blers. Both data structures are initially empty and are
populated as sequencing reads are processed in the
first stage. The de Bruijn graph is implemented with a
hash table, where k-mer graph edges are stored as keys
in the table and k-mer edge occurrences are stored as
values in the table. Graph nodes are stored implicitly. The Burrows-Wheeler index keeps track of assembled
candidate transcripts. For each sequencing read, its set
of k-mers is determined. If a k-mer already occurs in
the Burrows-Wheeler index, that is, is already part of
an assembled candidate transcript, then the k-mer is
not considered further. Materials and methods
Assembly algorithm As input, Rockhopper 2 requires one or more files of se-
quencing reads. Sequencing read files may be in fastq,
qseq, fasta, sam, or bam format [15]. Files in fastq, qseq,
or fasta format optionally may be gzipped. Rockhopper 2
works with single-end reads as well as paired-end reads,
and reads may be strand-specific or strand-ambiguous. A de Bruijn graph has 4k potential edges, which requires
more memory to store than is available on most personal
computers. As RNA-seq experiments continue to generate
increasing amounts of sequencing data, this limit will be
approached in de Bruijn graph-based assemblers, unless
sequencing error rates drop dramatically. Thus, most as-
semblers require high-performance computing hardware
with enhanced memory resources. Rockhopper 2 takes a
different approach and limits the size of de Bruijn graphs. Rockhopper 2’s approach has two main advantages: it en-
ables the system to run on common personal computers
and it quickly channels resources away from low fre-
quency k-mers that are likely to correspond to sequencing
errors or other artifacts. De novo transcriptome assembly in Rockhopper 2 pro-
ceeds in two stages (Figure 1). In the first stage, candi-
date transcripts are assembled from k-mers found in the
sequencing reads (k = 25 by default). After the first stage,
every k-mer in an assembled candidate transcript will
correspond to at least one k-mer from a sequencing
read. However, candidate transcripts may not be sup-
ported by full-length reads. Thus, in a second stage, se-
quencing reads are mapped to candidate transcripts in
order to filter candidate transcripts into a set of high
quality final transcripts that are well supported by full-
length sequencing reads. Algorithmic details of each
stage are provided below. In the first stage, we assemble candidate transcripts and
store them in a Burrows-Wheeler index. In the second
phase, we make a second pass through the sequencing
reads, aligning each read to the index. Importantly, the
Burrows-Wheeler index allows for rapid alignment in that
a read of length m can be aligned to the set of candidate
transcripts with total length N in O(m) time with small
constants. In the case of paired-end reads, we require that
the paired-ends for each read form a scaffold consistent In the first stage of de novo transcriptome assembly,
Rockhopper 2 maintains two data structures, a de Bruijn
graph [16,17] and a Burrows-Wheeler index [18,19]. Introduction If the k-mer is not part of an
assembled candidate transcript, then the de Bruijn
graph is updated with the k-mer. As k-mers are added to the de Bruijn graph, it grows
in size. As more memory is consumed and the amount
of available memory approaches zero, Rockhopper 2 re-
duces the size of the de Bruijn graph by assembling can-
didate transcripts. Paths through the graph are traversed,
beginning with the most frequently occurring edges. For
each edge with frequency at least α, a path is started and
greedily extended if a neighboring edge can be found
with frequency at least β (default values α = 50 and β = 5
were determined empirically). When an edge is traversed,
it is removed from the graph. A path corresponds to an
assembled candidate transcript. When a path is extended
as far as possible, the corresponding assembled candidate
transcript is added to the Burrows-Wheeler index. Materials and methods
Assembly algorithm While de Bruijn graphs are common among de novo
assemblers [4,17], Burrows-Wheeler indices are not. Instead, Burrows-Wheeler indices are common in
reference-based assemblers [2]. But it is precisely the Page 3 of 10 Page 3 of 10 Tjaden Genome Biology (2015) 16:1 Figure 1 Rockhopper 2 workflow depicting the various phases of Rockhopper 2’s analyses. As input, Rockhopper 2 requires one or more
files of sequencing reads from RNA-seq experiments. In the first analysis stage, Rockhopper 2 determines k-mers from the sequencing reads and
builds a de Bruijn graph from the k-mers. The de Bruijn graph is used to assemble candidate transcripts, which are stored in a Burrows-Wheeler
index. In the second analysis stage, Rockhopper 2 aligns the sequencing reads to the assembled candidate transcripts to determine a final set of
high-quality assembled transcripts. After the second stage, transcriptome assembly is complete and Rockhopper 2 performs several downstream
analyses, including normalizing data from different experiments, quantifying transcript abundance, and testing for differential gene expression
across multiple conditions. Figure 1 Rockhopper 2 workflow depicting the various phases of Rockhopper 2’s analyses. As input, Rockhopper 2 requires one or more
files of sequencing reads from RNA-seq experiments. In the first analysis stage, Rockhopper 2 determines k-mers from the sequencing reads and
builds a de Bruijn graph from the k-mers. The de Bruijn graph is used to assemble candidate transcripts, which are stored in a Burrows-Wheeler
index. In the second analysis stage, Rockhopper 2 aligns the sequencing reads to the assembled candidate transcripts to determine a final set of
high-quality assembled transcripts. After the second stage, transcriptome assembly is complete and Rockhopper 2 performs several downstream
analyses, including normalizing data from different experiments, quantifying transcript abundance, and testing for differential gene expression
across multiple conditions. Figure 1 Rockhopper 2 workflow depicting the various phases of Rockhopper 2’s analyses. As input, Rockhopper 2 requires one or more
files of sequencing reads from RNA-seq experiments. In the first analysis stage, Rockhopper 2 determines k-mers from the sequencing reads and
builds a de Bruijn graph from the k-mers. The de Bruijn graph is used to assemble candidate transcripts, which are stored in a Burrows-Wheeler
index. In the second analysis stage, Rockhopper 2 aligns the sequencing reads to the assembled candidate transcripts to determine a final set of
high-quality assembled transcripts. Materials and methods
Assembly algorithm After the second stage, transcriptome assembly is complete and Rockhopper 2 performs several downstream
analyses, including normalizing data from different experiments, quantifying transcript abundance, and testing for differential gene expression
across multiple conditions. Following de novo assembly of high quality transcripts,
Rockhopper 2 proceeds with several post-assembly phases
of analysis. To enable comparison between different
samples and experiments, Rockhopper 2 normalizes
each RNA-seq data set using upper quartile normalization
[20]. Transcript abundance levels are estimated using a Following de novo assembly of high quality transcripts,
Rockhopper 2 proceeds with several post-assembly phases
of analysis. To enable comparison between different
samples and experiments, Rockhopper 2 normalizes
each RNA-seq data set using upper quartile normalization
[20]. Transcript abundance levels are estimated using a with the transcript. We keep track of how many full-
length reads align to each candidate transcript and at
what loci. Sufficiently long regions of candidate tran-
scripts are retained as high quality finalized transcripts
if at least ε reads align throughout the length of the re-
gion (ε = 20 by default). Page 4 of 10 Tjaden Genome Biology (2015) 16:1 Tjaden Genome Biology (2015) 16:1 measure similar to RPKM (reads per kilobase per million),
which sums the number of reads for a transcript and
divides by the transcript’s length and a normalization
factor [7]. While the total number of reads in the sam-
ple is often used to determine the RPKM normalization
factor, Rockhopper 2 uses the more robust normalizer
of upper quartile transcript expression [20]. Finally,
Rockhopper 2 tests for differential transcript expression
in pairs of conditions using the algorithm of DESeq
[21]. In summary, Rockhopper 2 estimates the variance
of a transcript’s expression, uses local regression to
obtain a smooth estimate of the variance, and then per-
forms a statistical test to determine whether a transcript
shows differential expression in data from two or more
conditions. The negative binomial distribution is used
as the statistical model in order to compute a P-value
indicating the probability of observing the transcript’s
expression levels in the different conditions by chance. To correct for multiple tests across the set of transcripts,
P-values are corrected and q-values are reported that con-
trol the false discovery rate using the Benjamini-Hochberg
procedure [22]. Kits (KAPA Biosystems (Wilmington, MA, USA)). Performance evaluation Performance evaluation
In order to evaluate Rockhopper 2’s performance, we
compared it with two leading de novo transcriptome as-
semblers:
Trinity
version
trinityrnaseq_r20140413p1
[8,25] and SOAPdenovo2 version 2.04 [10,26]. Default
parameters were used for Trinity (Trinity –seqType fq -JM
10G -CPU 8) and SOAPdenovo2 (SOAPdenovo-63mer
all -p 8 -d 49). All three assemblers were executed on the
same hardware with the number of processors set to 8. The three software systems were used to assemble tran-
scriptomes using sequencing data from 12 microorganisms Materials and methods
Assembly algorithm The li-
braries were quantified by quantitative PCR , pooled in
equimolar concentration, and sequenced on one lane for
101 cycles from one end of the fragments using a TruSeq
SBS version 3 sequencing kit (Illumina (San Diego, CA,
USA)). The fastq files were generated with Casava 1.8.2
(Illumina). RNA-seq data from E. coli, Streptococcus pyogenes,
Mycobacterium tuberculosis, Bacillus subtilis, Staphylococ-
cus aureus, Pyrococcus abyssi, Acinetobacter oleivorans,
Propionibacterium acnes, Methanobrevibacter smithii,
Clostridium acetobutylicum, and Deinococcus gobiensis
were downloaded from the Sequence Read Archive
(SRA) [23]. Details on each RNA-seq data set, including
accession number in the SRA, length of the reads,
whether the reads are single-end or paired-end, and the
number of reads, is provided in Table 1. The Schizosac-
charomyces pombe RNA-seq data [24] were downloaded
from the Trinity tutorial [25]. High-throughput sequencing data Since not all genes are likely to be
expressed in a given experiment, the de novo assembled
transcripts are compared not against all annotated genes
but against a subset of annotated genes, which we call ref-
erence genes. A reference gene is defined as a gene where
every k-mer in the gene sequence (k = 25) occurs in at
least one sequencing read. Reference genes can possibly
be reconstructed by the de novo assemblers whereas non-
reference genes cannot. The set of reference genes is
analogous to the Oracle Set used to evaluate the Trinity
system [8]. X
g∈RI
gT0
gj j ≥δ
R
j j X
g∈RI
gT0
gj j ≥δ
R
j j X
g∈RI
gT0
gj j ≥δ
R
j j where gT
0
is the number of nucleotides in the sequence of
reference gene g that are covered via alignment by the
transcript from T that has the longest alignment to g. I is
an indicator function with parameter δ set to 0.8. RMBT (reads mapping back to transcripts) represents
the percentage of sequencing reads that align to an as-
sembled transcript. RMBT is given by: Specificity is a measure that represents the percentage
of assembled transcripts that align to the genome. Specifi-
city can be expressed as (1.0 - False positive rate), where a
false positive is an assembled transcript that does not align
to the genome. Specificity is calculated as: X
s∈SI
as
T
j j
sj j ≥δ
Sj j X
s∈SI
as
T
j j
sj j ≥δ
Sj j where S is the set of sequencing reads, as
T is the align-
ment of the read s to the set of assembled transcripts T,
and as
T
is the length of the alignment. I is an indicator
function with parameter δ set to 1.0. X
t∈TI
at
G
j j
tj j ≥δ
T
j j T
j j Accuracy represents the percentage of correctly as-
sembled bases; that is, for those transcripts that align to
the genome, the accuracy is the percentage of perfect
matches in the alignments (as opposed to mismatches or
gaps). Accuracy is calculated as: where T is the set of assembled transcripts and G is a
genome. High-throughput sequencing data Escherichia coli strain MG1655 was used in three biological
replicate DNA-seq experiments (Cari Vanderpool, personal
communication). Library construction and sequencing on
an Illumina HiSeq 2500 were performed at the WM Keck
Center for Comparative and Functional Genomics at the
University of Illinois at Urbana-Champaign. The DNA li-
braries were prepared with the KAPA Library Preparation Table 1 Sequencing data sets
Organism
Type
Domain
Class
SRA accession
number
Read type
Length of
reads (bp)
Number
of reads
Number of
reference
genes
Escherichia coli
DNA-seq
Bacteria
Gammaproteobacteria
SRP049375
Single
100
67,713,365
-
Escherichia coli
RNA-seq
Bacteria
Gammaproteobacteria
SRX254784
Single
100
34,085,732
4,190
Acinetobacter oleivorans
RNA-seq
Bacteria
Gammaproteobacteria
SRX560107
Paired
101
19,140,537
2,934
Deinococcus gobiensis
RNA-seq
Bacteria
Deinococci
SRX061110
Paired
75
18,676,333
610
Mycobacterium tuberculosis
RNA-seq
Bacteria
Actinobacteria
SRX380298
Paired
51
2,364,009
752
Streptococcus pyogenes
RNA-seq
Bacteria
Bacilli
SRX252449
Single
72
7,049,947
372
Bacillus subtilis
RNA-seq
Bacteria
Bacilli
SRX533166
Single
51
14,010,827
1,917
Staphylococcus aureus
RNA-seq
Bacteria
Bacilli
SRX172891
Paired
101
9,067,797
1,720
Propionibacterium acnes
RNA-seq
Bacteria
Actinobacteria
SRX278003
Single
75
195,541,304
1,777
Clostridium acetobutylicum
RNA-seq
Bacteria
Clostridia
SRX316281
Single
50
13,256,052
202
Pyrococcus abyssi
RNA-seq
Archaea
Thermococci
SRX556571
Single
40
51,342,770
133
Methanobrevibacter smithii
RNA-seq
Archaea
Methanobacteria
SRX031877
Single
36
32,744,832
211
Schizosaccharomyces pombe
RNA-seq
Eukarya
Schizosaccharomycetes
NA
Paired
68
4,000,000
3,591
The table summarizes the DNA-seq data set and the 12 RNA-seq data sets used in this study. Information in the table includes the length and number of sequencing
reads in each data set. NA, not available. Tjaden Genome Biology (2015) 16:1 Page 5 of 10 rather than from reference genes, covered by assembled
transcripts: with sequenced and annotated genomes, though the
genomes and their annotations were not used by any of
the software systems during assembly. The genome se-
quences and annotations were used only to evaluate
the de novo assembled transcriptomes. GT
= G
j j Contiguity represents the percentage of reference genes
that are at least δ = 80% covered by a single longest assem-
bled transcript [4]. Contiguity is defined as: A variety of measures was used to evaluate the perform-
ance of the different assemblers [4,27]. In some cases, the
correspondence between assembled transcripts and anno-
tated genes is assessed. High-throughput sequencing data at
G is the alignment of the sequence of t to
the sequence of G, and at
G
is the length of the align-
ment. I is an indicator function with parameter δ set
to 1.0. X
t∈TPM at
G
X
t∈T at
G
Sensitivity represents the percentage of sequence
from reference genes covered by assembled transcripts. Sensitivity in this context is sometimes referred to as
the coverage or completeness of an assembler. Sensitivity
is given by: Sensitivity represents the percentage of sequence
from reference genes covered by assembled transcripts. Sensitivity in this context is sometimes referred to as
the coverage or completeness of an assembler. Sensitivity
is given by: where PM at
G
is the number of perfect matches in the
alignment of the sequence t to the sequence of G, and
at
G
is the length of the alignment. Efficiency represents
the execution time of an assembler, as measured in mi-
nutes. Resourcefulness represents the amount of memory
(RAM) required during an assembly. X
g∈R gT
X
g∈R gj j X
g∈R gT
X
g∈R gj j gj j Results Further, Rockhopper 2’s as-
sembly had a sensitivity of approximately 90%, indicating
that 90% of the genome was covered by Rockhopper 2’s
assembled contigs (Figure 2B). While Rockhopper 2 was
not designed as a genome assembler, these results suggest
that it does a plausible job of reconstructing most of the
E. coli genome. For comparison, the contigs assembled
by SOAPdenovo2 and Trinity covered just under half
the E. coli genome (Figure 2B). Regions of the genome
that were not covered by contigs from any of the as-
semblers generally correspond to some combination of
repeat regions, errors in sequencing reads, and biases
during library construction and high-throughput sequen-
cing. Finally, we found that Rockhopper 2 required about
32 minutes to generate its assembly, a rate comparable to and the genome sequences and annotations. To provide
points of comparison, two leading de novo transcriptome
assemblers, Trinity [8,25] and SOAPdenovo2 [10,26],
were executed on the same data, and their results are
compared with those of Rockhopper 2. Results where R is the set of reference genes. Following align-
ment of assembled transcripts in T to the set of refer-
ence genes R, |gT| is the number of nucleotides in the
sequence of reference gene g that are covered via align-
ment by one or more transcripts from T. In the special
case of assessing the quality of assemblies from DNA-
seq data rather than RNA-seq data, sensitivity represents
the percentage of sequence from the entire genome, In order to assess Rockhopper 2’s ability to assemble
transcriptomes de novo, we executed Rockhopper 2 on
high-throughput sequencing data from a variety of or-
ganisms whose genomes have been sequenced and anno-
tated. The genome sequences and annotations were not
used by Rockhopper 2, rather they allow us to evaluate
Rockhopper 2 by investigating the correspondence be-
tween the de novo assembled transcriptomes it produces Page 6 of 10 Tjaden Genome Biology (2015) 16:1 SOAPdenovo2 both had close to 100% specificity, indicat-
ing that the vast majority of their assembled contigs could
be aligned to the E. coli genome (Figure 2A). In contrast,
just over half of the contigs assembled by Trinity were
considered false positives in that they did not align to the
E. coli genome (Figure 2A). Further, Rockhopper 2’s as-
sembly had a sensitivity of approximately 90%, indicating
that 90% of the genome was covered by Rockhopper 2’s
assembled contigs (Figure 2B). While Rockhopper 2 was
not designed as a genome assembler, these results suggest
that it does a plausible job of reconstructing most of the
E. coli genome. For comparison, the contigs assembled
by SOAPdenovo2 and Trinity covered just under half
the E. coli genome (Figure 2B). Regions of the genome
that were not covered by contigs from any of the as-
semblers generally correspond to some combination of
repeat regions, errors in sequencing reads, and biases
during library construction and high-throughput sequen-
cing. Finally, we found that Rockhopper 2 required about
32 minutes to generate its assembly, a rate comparable to SOAPdenovo2 both had close to 100% specificity, indicat-
ing that the vast majority of their assembled contigs could
be aligned to the E. coli genome (Figure 2A). In contrast,
just over half of the contigs assembled by Trinity were
considered false positives in that they did not align to the
E. coli genome (Figure 2A). Genomic DNA-seq data We used three biological replicates of genomic DNA-seq
data from E. coli (see Materials and methods) for pre-
liminary assessment of Rockhopper 2’s performance. Genome assembly based on DNA-seq data is generally
more
straightforward
than
transcriptome
assembly
based on RNA-seq data since RNA-seq data correspond
to transcripts with highly variable expression levels and
lengths, whereas DNA-seq data do not. Thus, the perform-
ance of an assembler using DNA-seq data can suggest an
upper bound on the quality of the assembly that we can ex-
pect from the assembler using RNA-seq data. Figure 2 and Additional file 1 provide statistics on as-
semblies based on the DNA-seq data. Rockhopper 2 and Figure 2 Performance assembling E. coli genome from DNA-seq data. The performance of Rockhopper 2 as well as two other assemblers,
SOAPdenovo2 and Trinity, on three biological replicate DNA-seq experiments from E. coli is illustrated. (A) Specificity is the percentage of assembled
contigs that align to the E. coli genome. (B) Sensitivity is the percentage of the E. coli genome sequence that is covered by assembled contigs aligning
to the genome. (C) Execution time is the number of minutes that an assembler requires to execute on the DNA-seq data. Figure 2 Performance assembling E. coli genome from DNA-seq data. The performance of Rockhopper 2 as well as two other assemblers,
SOAPdenovo2 and Trinity, on three biological replicate DNA-seq experiments from E. coli is illustrated. (A) Specificity is the percentage of assembled
contigs that align to the E. coli genome. (B) Sensitivity is the percentage of the E. coli genome sequence that is covered by assembled contigs aligning
to the genome. (C) Execution time is the number of minutes that an assembler requires to execute on the DNA-seq data. Page 7 of 10 Page 7 of 10 Tjaden Genome Biology (2015) 16:1 that of SOAPdenovo2 and substantially faster than that of
Trinity (Figure 2C). that of SOAPdenovo2 and substantially faster than that of
Trinity (Figure 2C). assumption that higher RMBT corresponds to a greater
percentage of reads used in constructing an assembly,
which is desirable in that it is more likely to lead to a ro-
bust assembly than using a smaller percentage of reads
when generating an assembly. Trinity and Rockhopper 2
consistently had high RMBT, in contrast to SOAPde-
novo2, suggesting that these two assemblers generally
use the majority of sequencing reads to construct their
assemblies (Figure 3D). RNA-seq data While DNA-seq data provide a starting point for under-
standing the quality of assemblies, the performance of
an assembler using RNA-seq data is more meaningful. Thus, we gathered data from RNA-seq experiments con-
ducted by 12 different labs for 12 different microorganisms
(see Materials and methods). The organisms included nine
bacteria, two archaea, and one fungus. While Rockhopper
2 was not designed for eukaryotic transcriptome assembly,
we evaluated its performance on data from the fungus S. pombe primarily because this same data set was used by
the authors of the Trinity assembler to assess Trinity’s per-
formance [8,25]. The 12 organisms represented in our ana-
lysis were chosen to reflect a wide range of phylogenetic
diversity in order to help us understand the robustness
of our assemblies. The 12 RNA-seq data sets range in
size from approximately 2 million reads to 200 million
reads and include 7 sets of single-end sequencing reads
and 5 sets of paired-end sequencing reads (Table 1). y
g
In order to investigate how Rockhopper 2’s assemblies
are affected by expression level, we evaluated Rockhopper
2’s sensitivity and contiguity across the 12 RNA-seq data
sets at different expression deciles (Figure 4). Each point
represents an average across the 12 data sets (Figure 4). For example, the leftmost point corresponds to the aver-
age sensitivity (purple) or contiguity (yellow) of Rockhopper
2’s assemblies across the 12 data sets for the 10% least
highly expressed reference genes. The rightmost point
corresponds to the average sensitivity (purple) or con-
tiguity (yellow) of Rockhopper 2’s assemblies across the
12 data sets for the 10% most highly expressed reference
genes. For this analysis, rather than requiring assembled
transcripts to align exactly to reference genes, we used a
more permissive alignment criterion and allowed assem-
bled transcripts to align to reference genes with a small
number of gaps or mismatches (BLAST E-value <0.01). Unsurprisingly, Rockhopper 2 is better able, in terms of
sensitivity and contiguity, to assemble transcripts with
higher expression than lower expression, as performance
generally improves as the expression decile increases
(Figure 4). However, there is a small decrease in perform-
ance at the very highest expression deciles (Figure 4). RNA-seq data This
asymmetric rainbow-shaped curve is consistent with what
others have observed [27], namely that assembly perform-
ance generally improves rapidly from the lowest expres-
sion quantiles to mid-level expression quantiles, plateaus
across mid-level expression quantiles to higher-level ex-
pression quantiles, and decreases slightly at the highest
level expression quantiles. These results provide one indi-
cation as to how confident a user can be in Rockhopper
2’s assembled transcripts for transcripts expressed at dif-
ferent levels. g
A variety of statistics (see Materials and methods) was
used to evaluate the assemblies produced by Rockhopper
2, Trinity, and SOAPdenovo2 across the 12 RNA-seq data
sets and the results are provided in Figure 3 and Additional
file 1. Rockhopper 2 and SOAPdenovo2 generally had the
highest specificity, generating fewer false positive assem-
bled transcripts that did not align to the corresponding
genome (Figure 3A). Interestingly, Rockhopper 2’s specifi-
city was lowest among the three assemblers on the two ar-
chaea data sets, but otherwise was among the highest. Assembly of additional RNA-seq data sets from prokary-
otes beyond the 11 used in this study will help illuminate
whether Rockhopper 2’s higher specificity on bacterial
data and lower specificity on archaeal data, relative to the
other two assemblers, is a broad trend resulting from
biases toward certain domains or if it is an artifact of a
small sample size. In terms of sensitivity, Rockhopper 2
demonstrated the highest sensitivity of the three assem-
blers across the 12 data sets, with its assembled transcripts
covering a significantly larger percentage of reference
genes than those of the other two assemblers (Figure 3B). Contiguity reflects the percentage of reference genes cov-
ered by a single longest transcript and is a useful measure
for distinguishing whether an assembly contains tran-
scripts covering a gene with multiple short transcripts or a
single long transcript. Rockhopper 2’s assemblies demon-
strate greater contiguity than those of the other two as-
semblers for 11 of the 12 data sets, with Trinity’s assembly
demonstrating the greatest contiguity for the S. pyogenes
data set (Figure 3C). RMBT indicates the percentage of
sequencing reads that align to assembled transcripts; this
measure is often used to evaluate assemblers under the We also considered a couple of additional measures of
assembly performance. Genomic DNA-seq data Finally, the execution time of
the assemblers was assessed. Both SOAPdenovo2 and
Rockhopper 2 demonstrated substantially greater efficiency
than Trinity across the 12 data sets (Figure 3E). RNA-seq data Accuracy is the percentage of cor-
rectly assembled bases and is computed by aligning as-
sembled transcripts to the corresponding genome and
calculating, for those transcripts that align, the percentage
of bases in the alignment that correspond to perfect
matches as opposed to mismatches or gaps [4]. All three
assemblers showed accuracies greater than 99% across all
12 RNA-seq data sets (Additional file 1). Resourcefulness Page 8 of 10 Tjaden Genome Biology (2015) 16:1 Figure 3 Performance assembling transcripts from RNA-seq data. The performance of each of three assemblers on 12 RNA-seq data sets is
illustrated. The 12 RNA-seq data sets correspond to nine bacteria, two archaea, and one fungus. (A) Specificity is the percentage of assembled
transcripts that align to the genome. (B) Sensitivity is the percentage of the reference gene sequences that is covered by assembled transcripts
aligning to the reference genes. (C) Contiguity is the percentage of reference genes that are at least δ = 80% covered by their single longest
aligning transcript. (D) RMBT is the percentage of sequencing reads to align to assembled transcripts. (E) Execution time is the number of minutes
that an assembler requires to execute on the RNA-seq data set. Figure 3 Performance assembling transcripts from RNA-seq data. The performance of each of three assemblers on 12 RNA-seq data sets is
illustrated. The 12 RNA-seq data sets correspond to nine bacteria, two archaea, and one fungus. (A) Specificity is the percentage of assembled
transcripts that align to the genome. (B) Sensitivity is the percentage of the reference gene sequences that is covered by assembled transcripts
aligning to the reference genes. (C) Contiguity is the percentage of reference genes that are at least δ = 80% covered by their single longest
aligning transcript. (D) RMBT is the percentage of sequencing reads to align to assembled transcripts. (E) Execution time is the number of minutes
that an assembler requires to execute on the RNA-seq data set. Figure 3 Performance assembling transcripts from RNA-seq data. The performance of each of three assemblers on 12 RNA-seq data sets is
illustrated. The 12 RNA-seq data sets correspond to nine bacteria, two archaea, and one fungus. (A) Specificity is the percentage of assembled
transcripts that align to the genome. (B) Sensitivity is the percentage of the reference gene sequences that is covered by assembled transcripts
aligning to the reference genes. RNA-seq data (C) Contiguity is the percentage of reference genes that are at least δ = 80% covered by their single longest
aligning transcript. (D) RMBT is the percentage of sequencing reads to align to assembled transcripts. (E) Execution time is the number of minutes
that an assembler requires to execute on the RNA-seq data set. Page 9 of 10 Tjaden Genome Biology (2015) 16:1 Figure 4 Rockhopper 2 performance at different expression
deciles. For each of the 12 RNA-seq data sets, the set of reference
genes was divided into 10 groups based on their expression levels,
with the 10% of reference genes with lowest expression in the first
group and the 10% of reference genes with highest expression in
the last group. The sensitivity (purple) and contiguity (yellow) of
Rockhopper 2’s assemblies across all 12 RNA-seq data sets
are illustrated. The algorithms have been implemented in an open-source
software system called Rockhopper 2. We evaluated Rockhopper 2 using one set of DNA-seq
data and 12 sets of RNA-seq data corresponding to a range
of microorganisms. We found that Rockhopper 2 produced
high quality transcriptome assemblies and outperformed
other leading assemblers. Rockhopper 2 has several other
advantageous features, including a graphical interface and
the ability to run on common laptops rather than necessi-
tating a high-performance computing environment. In
addition to de novo transcriptome assembly, the Rockhopper
2 system integrates algorithms for normalization of
data across experiments, quantification of transcript
abundance, and testing for differential gene expression. Thus, Rockhopper 2 reduces the initial stages of ana-
lysis of large bacterial RNA-seq data sets to a matter of
minutes, enabling investigators to spend more time on
downstream interpretation of results and extraction of
new biological insights. Figure 4 Rockhopper 2 performance at different expression Figure 4 Rockhopper 2 performance at different expression
deciles. For each of the 12 RNA-seq data sets, the set of reference
genes was divided into 10 groups based on their expression levels,
with the 10% of reference genes with lowest expression in the first
group and the 10% of reference genes with highest expression in
the last group. The sensitivity (purple) and contiguity (yellow) of
Rockhopper 2’s assemblies across all 12 RNA-seq data sets
are illustrated. Abbreviations bp: base pair; PCR: polymerase chain reaction; RAM: random-access memory;
RMBT: reads mapping back to transcripts; RPKM: reads per kilobase per
million; SRA: Sequence Read Archive. Conclusions Transcriptome assembly is a common step in the analysis
of RNA-seq data. When a sequenced genome is available,
assembly approaches can leverage the reference genome
by aligning sequencing reads to the genome. When a
high-quality reference genome is not available, transcrip-
tomes must be assembled de novo. While a number of ma-
ture tools exist for de novo assembly of transcriptomes
from RNA-seq data, these tools were designed primarily
for eukaryotic data and their performance suffers when
applied to bacterial data. In this study, we propose novel al-
gorithms for de novo assembly of bacterial transcriptomes. Author’s contributions BT designed the algorithms, implemented the software, and wrote the
manuscript. Received: 20 August 2014 Accepted: 15 December 2014 Received: 20 August 2014 Accepted: 15 December 2014 Received: 20 August 2014 Accepted: 15 December 2014 Additional file reflects the amount of RAM consumed during an assem-
bly. A comparison of memory usage by SOAPdenovo2
and Trinity has been performed by others [27] and we did
not repeat the analysis here. With default parameters,
Rockhopper 2 uses at most 1.2 GB (gigabytes) of memory. For the assemblies in this study, we allowed Rockhopper 2
to use up to 2.0 GB of memory, an amount generally avail-
able on any common laptop. In contrast, the authors of
the Trinity assembler recommend approximately 1 GB of
memory per million paired reads for Trinity [25]. The
RNA-seq data sets used in this study contained between 2
million and 195 million reads, with an average of 36 mil-
lion reads. Thus, Trinity’s memory consumption typically
requires high-performance computing hardware whereas
Rockhopper 2 has no such requirement. All three assem-
blers are fully parallelizable and their runtime performance
scales inversely with the number of processors available
for computation in the machine on which the assembler
is executed. Additional file 1: For the DNA-seq data set and the 12 RNA-seq data
sets used in this study, the table provides details on the performance
statistics for each of three assemblers when generating assemblies
from each data set. Additional file 1: For the DNA-seq data set and the 12 RNA-seq data
sets used in this study, the table provides details on the performance
statistics for each of three assemblers when generating assemblies
from each data set. The author declares that he has no competing interests. The author declares that he has no competing interests. Acknowledgements This work was supported by the National Institutes of Health grant R15
GM102755 to BT. The author would like to thank Cari Vanderpool and Alisa
King for genomic DNA sample preparation and sequencing. The author
would also like to thank members of the Rockhopper and Rockhopper 2
user community for their valuable feedback on the software systems. References 1. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomes. Nat Rev Genet. 2009;10:57–63. 1. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomes. Nat Rev Genet. 2009;10:57–63. 2. Garber M, Grabherr MG, Guttman M, Trapnell C. Computational methods for
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study. BMC Bioinformatics. 2011;12:S2. References Submit your next manuscript to BioMed Central
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https://openalex.org/W2162072605
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https://journals.uni-lj.si/DocumentaPraehistorica/article/download/36.8/1771
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English
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Demographic model of the Neolithic transition in Central Europe
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Documenta Praehistorica
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cc-by-sa
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UDK 903ı12\ı15(292.45)"633\634">314.18
Documenta Praehistorica XXXVI (2009) UDK 903ı12\ı15(292.45)"633\634">314.18
Documenta Praehistorica XXXVI (2009) Documenta Praehistorica XXXVI (2009) Patrik Galeta 1, Jaroslav Bruzek 2
1 Department of Anthropology, University of West Bohemia, Pilsen, CZ
galeta@ksa.zcu.cz
2 UMR 5199, PACEA, Laboratoire d’Anthropologie des Populations du Passé, Université Bordeaux I, Talence, FR
Department of Anthropology, Charles University, Prague, CZ Patrik Galeta 1, Jaroslav Bruzek 2
1 Department of Anthropology, University of West Bohemia, Pilsen, CZ
galeta@ksa.zcu.cz
2 UMR 5199, PACEA, Laboratoire d’Anthropologie des Populations du Passé, Université Bordeaux I, Talence, FR
Department of Anthropology, Charles University, Prague, CZ ABSTRACT – Several recent lines of evidence indicate more intensive contact between LBK farmers
and indigenous foragers in Central Europe (5600–5400 calBC). Strong continuity has been identified
between Mesolithic and Neolithic material cultures; faunal assemblages, and isotopic analyses of diet
have revealed a greater role of hunting in LBK communities; genetic analyses have suggested that the
modern Central European gene pool is mainly of Palaeolithic origin. Surprisingly little attention has
been paid to demographic aspects of the Neolithic transition. In our study, demographic simulations
were performed to assess the demographic conditions that would allow LBK farmers to spread across
central Europe without any admixture with Mesolithic foragers. We constructed a stochastic demogra-
phic model of changes in farming population size. Model parameters were constrained by data from
human demography, archaeology, and human ecology. Our results indicate that the establishment of
farming communities in Central Europe without an admixture with foragers was highly improbable. The demographic conditions necessary for colonization were beyond the potential of the Neolithic po-
pulation. Our study supports the integrationists’ view of the Neolithic transition in Central Europe. IZVLE∞EK – Ve≠ novih dokaznih linij ka∫e na intenzivnej∏e stike med LKB poljedelci in prvotnimi na-
biralci v srednji Evropi (5700–5500 calBC). Dognana je bila mo∫na kontinuiteta med mezolitskimi
in neolitskimi materialnimi kulturami; favnisti≠ni zbiri in izotopske analize prehrane ka∫ejo ve≠jo
vlogo lova v LBK skupnostih; genetske analize ka∫ejo, da je moderni srednje evropski genetski fond
prete∫no paleolitskega izvora. Presenetljivo malo pozornosti je bilo posve≠ene demografskim aspek-
tom neolitizacije. V na∏i ∏tudiji uporabljamo demografske simulacije, da bi ocenili demografske po-
goje, ki bi omo≠ili LKB poljedelcem ∏iritev preko srednje Evrope brez kakr∏negakoli me∏anja z mezo-
litskimi nabiralci. Oblikovali smo stohasti≠ni demografski model sprememb v velikosti poljedelske po-
pulacije. Parametri modela so bili izvedeni iz podatkov o humani demografiji, arheologiji in huma-
ni ekologiji. Na∏i rezultati ka∫ejo, da je bila ustanovitev poljedelskih skupnosti brez me∏anja z nabi-
ralci malo verjetna. Demografski pogoji potrebni za kolonizacijo so presegali potencial neolitske po-
pulacije. Na∏a ∏tudija podpira integracionisti≠ni pogled na neolitizacijo v srednji Evropi. KEY WORDS – demographic simulations; Neolithic transition; Central Europe; colonization; fertil-
ity; population growth KEY WORDS – demographic simulations; Neolithic transition; Central Europe; colonization; fertil-
ity; population growth Introduction tribution of Near Eastern farmers and indigenous for-
agers to the establishment of farming communities. Three alternative explanations of the spread of agri-
culture across Europe have been proposed, which
were summarized by Zvelebil (2000) as the migra-
tionist, indigenist, and integrationist positions. Migra- The pattern of the introduction of domesticated
plants and animals into Europe has been a subject
of major interest for more than one hundred years
(Gronenborn 2007). Although it is generally accep-
ted that farming spread into Europe from the Near
East, disagreements prevail about the relative con- 139 DOI: 10.4312/dp.36.8 Patrik Galeta, Jaroslav Bruzek Fig. 1. Map of the LBK origin area in Western Hungary (dark grey) and
the area settled after the Earliest LBK expansion over Central Europe
from 5600 to 5400 calBC (light grey). Adapted from Zvelebil (2001.Fig. 2). tionists favor the spread of
farmers, with the genetic re-
placement of Mesolithic for-
agers; indigenists prefer the
spread of farming with no ge-
netic contribution from the
Near East; and integrationists
emphasize both people and
ideas, and presume a genetic
admixture of foragers and far-
mers. Recently, it has become clear
that the spread of agriculture
across Europe cannot be mo-
deled monocausally. The
spread involved a variety of
mechanisms that were shaped
by regional conditions. On
the one hand, local Mesolithic
groups played a significant
role in the spread of agricul-
ture throughout much of
Northern Europe, the Alps,
the Atlantic fringe of France and Central Iberia. On
the other hand, the Eastern Mediterranean and
South-Eastern Europe are regions that probably ex-
perienced farmer migration (Zvelebil 2000; Robb
and Miracle 2007). Similarly, the spread of farming
across Central Europe has traditionally been accep-
ted as an example of agricultural colonization by far-
mers of Linear Pottery Culture (LBK) (Childe 1925;
Piggott 1965; Vencl 1986; Lüning 1988; Price et al. 1995; Bogucki 2001; Neustupný 2004). It is belie-
ved, that LBK farmers spread within 4–6 generati-
ons from its origin in Western Hungary over the
broad area extending from Western Ukraine to the
Rhine River in Germany (Fig. 1). Recently, the migra-
tionist view that the LBK spread across Central Eu-
rope has been challenged and, today, the integratio-
nist view is accepted by the majority of scholars from
continental Europe concerned with the Central Early
Neolithic (Gronenborn 2007). Fig. 1. Introduction 2005; Davison et al. 2006; Davison et al. 2007). pulation dynamics, such as the wave of advance mo-
del (Ammerman and Cavalli-Sforza 1973) and its
various generalizations (Fort and Mendéz 1999; Pin-
hasi et al. 2005; Davison et al. 2006; Davison et al. 2007). meters, we applied a simple mathematical solution. We modified the basic exponential equation Pt =
P0ert (Newell 1988.182), where P0 and Pt are pop-
ulation size at the beginning and end of the expan-
sion; t is the duration of the process in years, and r
is the growth rate. The exponential curve is presen-
ted in Fig. 2. It may be seen that population size (Y
axis) is a function of only two parameters: time (X
axis) and growth rate (the slope of the curve). meters, we applied a simple mathematical solution. We modified the basic exponential equation Pt =
P0ert (Newell 1988.182), where P0 and Pt are pop-
ulation size at the beginning and end of the expan-
sion; t is the duration of the process in years, and r
is the growth rate. The exponential curve is presen-
ted in Fig. 2. It may be seen that population size (Y
axis) is a function of only two parameters: time (X
axis) and growth rate (the slope of the curve). So far, there have been only a few attempts to esti-
mate the growth and/or fertility rates of the LBK
population directly from the archaeological evidence. Neustupný (1983) produced abridged life tables from
LBK skeleton samples from eastern Germany and
estimated the growth rate at 1–2%. A similar value
was calculated by Petrasch (2001). His analysis was
based on the function of exponential growth, and
input variables were derived from the distribution
of LBK settlements and radiocarbon data. Unfortuna-
tely, both estimates are deterministic, and do not ac-
count for the uncertainty associated with adopting
input parameters from archaeological sources. We rearranged the basic equation describing expo-
nential growth to obtain the growth rate r = ln(Pt/P0)
/t. Because we were not able to estimate the LBK
population size with sufficient accuracy, we replaced
it with the product of area size and population den-
sity. Introduction The last equation was then rewritten as r =
ln(At.d/A0)/t, where A0 and At are the size of the ori-
gin area and settled area respectively, and d is the
ratio of population density at the end to density at
the beginning of the expansion (d = dt/d0). In this study, we built a stochastic demographic mo-
del that describes the demographic conditions of
Neolithic transition in Central Europe. Demographic
simulations were performed to directly test the col-
onization hypothesis. In particular, our question is
whether the growth and fertility rates of Earliest
LBK population could have been high enough to
allow the farmers to colonize Central Europe with-
out mixing with the local Mesolithic foragers. We then used the estimate of growth rate to mea-
sure LBK fertility. Total fertility rate (TFR), which is
the number of children born to average woman, was
calculated according to the equation: TFR = er·g/S.lg
(Hinde 2002.25), where g is the generation length,
lg is the proportion of females surviving to g years
of age, and S is the proportion of females at birth. Introduction Map of the LB
the area settled aft
from 5600 to 5400 c Fig. 1. Map of the LBK origin area in Western Hungary (dark grey) and
the area settled after the Earliest LBK expansion over Central Europe
from 5600 to 5400 calBC (light grey). Adapted from Zvelebil (2001.Fig. 2). German have demonstrated relatively high stable ni-
trogen ratio values, traditionally interpreted as a re-
liance on animal protein (Dürrwächter et al. 2006). Several authors have suggested that late Mesolithic
foragers practiced some kind of small-scale farming
(Erny-Rodmann et al. 1997; Tinner et al. 2007). Strontium isotope analyses of human skeletons from
LBK cemeteries in South-Western Germany have re-
vealed a significant amount of non-locals, which
would indicate that foragers had joined LBK commu-
nities (Price et al. 2001; Bentley 2007). Genetic stu-
dies of the classical markers, mtDNA, and Y-chromo-
some, have indicated a significant contribution from
Mesolithic foragers to the gene pool of modern Eu-
ropeans (Richards 2003). The admixture view has
been strengthened by the direct extraction of mtDNA
from skeletons buried at LBK cemeteries in Germany
and Austria (Haak et al. 2005). Given the fact that many different disciplines have
been involved in explaining the mechanism of Neo-
lithic dispersal, it is surprising how little has been
done in the field of demography. Authors have only
generally presumed that the prerequisite of the colo-
nization would have been a high rate of population
growth (Crubézy et al. 2002), and LBK farmers would
have had to reproduce at the rate approaching the
theoretical maximum for human population. A
growth rate of from 2.0% to 3.5% per year has been
universally used as the input value in models of po- The integrationist position is supported by a number
of indicators of contact between foragers and farm-
ers. Typological and technological analyses of lithic
assemblages show a continuity in stone tool produc-
tion from the Mesolithic to the Earliest LBK (Gronen-
born 1998; Kind 1998). Some Earliest LBK sites yield
relatively high amounts of game, which might be in-
terpreted as an interaction between Earliest LBK and
Mesolithic groups (Gronenborn 1999). Also, stable
isotope analyses of LBK skeletons from Southern 140 Demographic model of the Neolithic transition in Central Europe pulation dynamics, such as the wave of advance mo-
del (Ammerman and Cavalli-Sforza 1973) and its
various generalizations (Fort and Mendéz 1999; Pin-
hasi et al. Demographic model Estimating female mortality was not straightforward. Women’s survival to the mean age at childbearing
(lg) was obtained from life tables. We did not rely
on the life tables of real prehistoric populations be-
cause the skeletal data that the tables stem from
were considered unreliable. Instead, we estimated
mortality from simulated life tables which we gene-
rated using the Brass two-parametric relational sys-
tem of model life tables (Brass 1971). The Brass lo-
git system is based on a generic survival function
which is transformed by a logit transformation into
a new survival curve. By varying either of two para-
meters, we generated 1000 model life tables with a
life expectancy at birth of between 18 and 25 years,
which is assumed to be the mortality level of the pre-
historic population (Gage 2005). Finally, women’s
survival to the mean age at
childbearing, the input para-
meter of our model, was ob-
tained from this set of simula-
ted life tables. Estimating female mortality was not straightforward. Women’s survival to the mean age at childbearing
(lg) was obtained from life tables. We did not rely
on the life tables of real prehistoric populations be-
cause the skeletal data that the tables stem from
were considered unreliable. Instead, we estimated
mortality from simulated life tables which we gene-
rated using the Brass two-parametric relational sys-
tem of model life tables (Brass 1971). The Brass lo-
git system is based on a generic survival function
which is transformed by a logit transformation into
a new survival curve. By varying either of two para-
meters, we generated 1000 model life tables with a
life expectancy at birth of between 18 and 25 years,
which is assumed to be the mortality level of the pre-
historic population (Gage 2005). Finally, women’s
survival to the mean age at
childbearing, the input para-
meter of our model, was ob-
tained from this set of simula-
ted life tables. The next three input parameters were acquired from
demographic sources. The relative proportion of fe-
males at birth (S) and mean age at childbearing (g)
have been assumed to be relatively stable among
human populations with natural reproduction (Hinde
2002). So we were able to find reliable point esti-
mates of both parameters. Demographic model In all simulations, the pro-
portion of females at birth was set to 0.4878 (100
females per 105 males) and mean age at childbea-
ring to 27.5 years of age. Also, density ratio (d) was
fixed in basic simulations to the single value of 100%,
which means that density was assumed to remain
constant during the spread of LBK. Fig. 3. Map showing the area settled by the Earliest LBK around 5400
calBC up to 350m above sea level. Base map adapted from Zvelebil (2001. Fig. 2). Demographic model The values of input and output parameters of the
model were obtained from archaeological, ethnogra-
phic and demographic sources. The list of input and
output parameters along with their values that were
entered in the simulations is presented in Table 1. In the following paragraphs we will explain in detail
the determination of these values. Our model is a demographic projection of the size
of the LBK population during the expansion across
Central Europe. To avoid estimations of many para- Fig. 2. Exponential growth. The function describes
how changes in population size (Y axis) depended
on time (X axis). Growth rate is reflected by the
steepness of the curve. The size of the LBK area of origin (A0) was compu-
ted in GIS software from four maps produced by ar-
chaeologists (Kalicz 1993; Petrasch 2001; Zvelebil
2001; Bánffy 2004). Similarly, the size of the area
settled during the expansion (At) was derived from
five maps of Earliest LBK site distribution (Lüning et
al. 1989; Gronenborn 1998; Bogucki 2000; Jochim
2000; Zvelebil and Lillie 2000). To avoid regions in
high altitudes we consider only part of the land-
scape up to 350m above sea level. This level was
suggested as an upper limit of LBK settlement acti-
vity. Only a small proportion of LBK settlements
have been discovered above the 350m contour (Rulf
1983; Květina 2001). Fig. 3 shows an example of the
area restricted by the 350m contour made for a map Fig. 2. Exponential growth. The function describes
how changes in population size (Y axis) depended
on time (X axis). Growth rate is reflected by the
steepness of the curve. 141 Patrik Galeta, Jaroslav Bruzek symbol description
n
min
max
A0
Origin area limited by 350 m a.s.l. contour ∂km2]
4
32.714
51.446
At
Settled area limited by 350 m a.s.l. contour ∂km2]
5
181,978 232.45
t
Time of spread ∂years]
12
100
200
g
Generation length ∂years]
8
27.5
27.5
S
Proportion of females at birth
5
0.4878 0.4878
lg
Proportion of females survived to the g years of age
1
0.24
0.43
d
Density ratio
1
100
100
TFR*
Total fertility rate ∂children per woman]
11
6.92
n> Number of estimates
Tab. 1. List of the input parameters and the output variable (*) of the de-
mographic model. Tab. 1. List of the input parameters and the output variable (*) of the de-
mographic model. Output variable and com-
parative sample of fertility The only output parameter of
our demographic model is a
measure of the fertility of the
LBK population, namely its
total fertility rate (TFR). To as-
sess the level of fertility ob-
tained in simulations, we crea-
ted the comparative sample
of TFR. The comparative sam-
ple comprises TFRs of eleven
recent populations with natu-
ral reproduction. Populations
included in the sample are
horticulturalists (extensive ag-
riculturalist) who cultivate ce- Fig. 3. Map showing the area settled by the Earliest LBK around 5400
calBC up to 350m above sea level. Base map adapted from Zvelebil (2001. Fig. 2). 142 Demographic model of the Neolithic transition in Central Europe reals and are sedentary. These characteristics have
traditionally been attributed to the LBK population
(Gregg 1988), although some authors have assumed
that LBK cultivators were familiar with some inten-
sive gardening techniques (Halstead 1989; Bogaard
2004). drawn at random from the interval shown in Table
1. These values were used to calculate the output va-
riable, i.e. TFR. Then, a process of random sampling
of input parameters and calculation of output vari-
able was run 10000 times. In the end, we obtained
10000 estimates of TFR. Each of the 10000 itera-
tions of the model represented one possible demo-
graphic scenario of the Neolithic transition in Cen-
tral Europe. drawn at random from the interval shown in Table
1. These values were used to calculate the output va-
riable, i.e. TFR. Then, a process of random sampling
of input parameters and calculation of output vari-
able was run 10000 times. In the end, we obtained
10000 estimates of TFR. Each of the 10000 itera-
tions of the model represented one possible demo-
graphic scenario of the Neolithic transition in Cen-
tral Europe. TFR data were gathered from two studies concer-
ned with the relationship between fertility and sub-
sistence (Bentley et al. 1993; Sellen and Mace 1997). The histogram of TFR in the comparative sample is
shown in Fig. 4. The distribution of TFR is highly
skewed to larger values. Populations with TFR grea-
ter than 6 prevail in the sample. The sample maxi-
mum is 6.7 children, but to obtain the parametric
maximum in the population (population in the sta-
tistical sense), we used an unbiased standard boot-
strap method of confidence limits calculation (Manly
2007). Output variable and com-
parative sample of fertility This parametric maximum we entitle here as
the critical value of TFR, and its value was calcula-
ted at 6.92 children born to the average woman. We
assumed that the critical value of TFR represents
the upper limit of fertility that could be attained by
LBK women during the Neolithic transition. Statistical and graphic analyses (descriptive statis-
tics, multivariate regression, randomization analy-
sis) were performed in MS Excel 2003 (© Microsoft
Corporation, 1985–2003) and STATISTICA 6.1 (©
StatSoft, 1984–2003). 3D surface charts were made
in R software (Ihaka and Gentleman 1996), version
2.8.0 (© 2008 The R Foundation for Statistical Com-
puting). Geographical data were analyzed in ArcMap
9.0 (© ESRI, 1999–2004). Results The descriptive statistics of 10000 estimates of TFR
and growth rate obtained in the simulations are
shown in Table 2. The growth rate of the farming
population ranges from 0.64% to 1.96% per year. The estimates of total fertility rates oscillate from
around 6 to 13 children per woman. The distribu-
tion of TFR is skewed (Fig. 6); lower values (up to 9
children) are more frequent in the simulations than
larger values. From both Table 2 and Fig. 6 it is evi-
dent that the majority of iterations give an estimate
of TFR greater than the critical value of fertility. In
fact, only 7.89% of TFR estimates are lower than the Randomization step Table 1 demonstrates that four out of seven input
variables are defined in range. Because we did not
want to reduce the interval estimates of input para-
meters only to a point estimate (e.g. average value),
we inserted a randomization step into the model. The randomization step is a stochastic component of
the simulations and is motivated by the complexity
associated with the input parameters. The principle
of the randomization step is described in Figure 5. First, a single value of each input parameter was Fig. 5. The principle of the demographic model
with a randomization component (see text for the
explanation). First, a single value of each input parameter was
Fig. 4. The distribution of total fertility rate (TFR)
in the comparative sample (n = 11 horticulture so-
cieties). Fig. 5. The principle of the demographic model
with a randomization component (see text for the
explanation). Th d
b
f
l f
l
T Fig. 4. The distribution of total fertility rate (TFR)
in the comparative sample (n = 11 horticulture so-
cieties). Fig. 5. The principle of the demographic model
with a randomization component (see text for the
explanation). 143 Patrik Galeta, Jaroslav Bruzek Fig. 6. The distribution of the output variable. Hi-
stogram comprises 10 000 estimates of TFR obtai-
ned in the simulations. Dashed line indicates the
position of the critical value of fertility for horticul-
tural societies (6.92 children, see text for explana-
tion). critical value of 6.92 children. In other words,
around 92% of the demographic scenarios of the
Neolithic transition in Central Europe contradict the
hypothesis of colonization. To assess the effect of input parameters, we perfor-
med a multiple regression analysis of data obtained
in 10000 iterations. As independent variables, we
selected only four input parameters that are defined
in range: origin area, settled area, time and survival
of females (see Tab. 1). The remaining input parame-
ters were excluded from the regression analysis be-
cause they were estimated by single values and have,
therefore, the same effect on TFR in each iteration. The analysis of residuals suggested that the regres-
sion trend in raw data is non-linear. To achieve linea-
rity, we transformed the raw data by natural loga-
rithm. The results of multivariate regression analy-
sis and basic statistics of ln transformed inputs para-
meters are given in Table 3. Multivariate regression
is highly significant (P<10–5). Randomization step The high value of the
coefficient of determination (0.996) indicates that
the regression provides a good fit to the data. In fact,
99.6% of the variability of TFR is explained by the
model. The standardized coefficients shown in Tab-
le 3 indicate that the greatest effects on TFR came
from the duration of spread and the survival of fe-
males. On the other hand, variation in the size of
the origin and settled area has minimal impact on
fertility estimate. Fig. 6. The distribution of the output variable. Hi-
stogram comprises 10 000 estimates of TFR obtai-
ned in the simulations. Dashed line indicates the
position of the critical value of fertility for horticul-
tural societies (6.92 children, see text for explana-
tion). nization hypothesis. The grey segments represent
demographic conditions that lead us to reject the co-
lonization hypothesis. For example, the combination
of 100% density, 40% survival and duration of 100
years (left contour graph) gives a TFR estimate of al-
most 8 children. Discussion 144 Demographic model of the Neolithic transition in Central Europe mean
V ∂%]
beta
SEbeta
origin area
10.6
1.2
–0.18
0.0006
settled area
12.2
0.6
0.10
0.0006
time
5.0
4.0
–0.46
0.0006
survival of females
–1.1
10.8
–0.87
0.0006
V> Coefficient of variation
beta> Standardized regression coefficient
SEbeta> Standard error of coefficient
Tab. 3. Effect of input parameters to output vari-
able (TFR). Regression analysis computed after ln
transformation. Coefficient of determination R2
= 99.6%. ly. Although some authors have shown that a human
population could have grown at a rate of around
3% in the past (Birdsell 1957), others have argued
that the development of agriculture negatively affec-
ted human health, led to poorer nutrition, and that
higher population density increased the probability
of transmission of infectious disease from livestock
to humans (Gage 2005). mean
V ∂%]
beta
SEbeta
origin area
10.6
1.2
–0.18
0.0006
settled area
12.2
0.6
0.10
0.0006
time
5.0
4.0
–0.46
0.0006
survival of females
–1.1
10.8
–0.87
0.0006
V> Coefficient of variation
beta> Standardized regression coefficient
SEbeta> Standard error of coefficient Similar results were obtained in the analysis of to-
tal fertility rate, which is the final parameter of the
demographic model. TFR vary approximately from
6 to 13 children (Tab. 2). Slightly more than 92% si-
mulations gave estimates of TFR greater than the
maximum level of fertility observed in the horticul-
ture populations (Tab. 2). Thus, it is more likely, that
LBK fertility was not high enough to allow farmers
to spread over Central Europe without admixture
with local foragers. Our demographic simulations
thus provide a strong argument against the hypoth-
esis of colonization. Tab. 3. Effect of input parameters to output vari-
able (TFR). Regression analysis computed after ln
transformation. Coefficient of determination R2
= 99.6%. stationary, i.e. with zero growth. Carneiro and Hilse
(1966) and Barringer (1966) have assumed that a
reasonable estimate of growth rate in the Neolithic
would be as high as 0.12% and 0.25% per year res-
pectively. Hassan and Sengel (1973) have estimated
that the average annual growth rate during the Neo-
lithic was about 0.1%. They suspected, however, that
growth rate would be uniform and it could, in fact,
attain values of 0.5–1.0% in a period of rapid popu-
lation increase. Discussion In this study, we estimated the level of fertility and
growth rate of the LBK population via demographic
modelling. The objective was to assess whether such
a level of fertility and growth rate could be high
enough to allow the LBK farmers to spread across
Central Europe within less than 200 years without
admixture with indigenous foragers. Although both
fertility and mortality levels can be estimated from
skeletal remains (Buikstra et al. 1986; Paine and
Harpending 1996; Bocquet-Appel 2002), the low
number of Earliest LBK cemeteries with well preser-
ved human remains and their non-random spatial
distribution restrict such attempts. In situations where
few empirical data are available, demographic simu-
lations are a powerful tool for answering similar que-
stions (cf. Steele et al. 1998; Alroy 2001; Surovell
2003). The relationship among total fertility rate and three
input parameters in the model is shown in Figure
7. The isolines in contour graphs connect points of
equal value of TFR. The ratio of population density
in the settled area to population density in the ori-
gin area is displayed on the X axis, and the propor-
tion of females surviving to 27.5 years of age on the
Y axis. The contour graph on the left shows the du-
ration of LBK initial spread through Central Europe
fixed to the value of 100 years, and to 200 years in
the graph on the right. Both contour graphs were
computed with average size of the origin and set-
tled area. The isoline at 6.92 children represents the
critical value of the total fertility rate of horticultu-
ral societies. The white parts of the graphs corre-
spond to the fertility estimates that match the colo- In this study, we estimated the growth rate range of
the LBK population at 0.64 to 1.96% per
year (Tab. 2). Comparison with the esti-
mates proposed by other authors suggests
that such values seem to be rather high for
the LBK population. Bocquet-Appel (2002)
has even estimated that the population un-
dergoing Neolithic transition in Europe was
92 ∂%]
9
growth
ons. Tab. 2. Descriptive statistics of 10 000 estimates of growth
rate and total fertility rate obtained in the simulations. Discussion Therefore, the actual level of
LBK fertility would have to be greater than we esti-
mated in the simulations. Thus, the colonization hy-
pothesis may be rejected with greater confidence. Therefore, a density value of 100% might be a rea-
sonable assumption in the simulations. It can be seen
in Figure 7 that there are some TFR estimates below
the threshold value of 6.92 children at the density
level of 100%. However, they can be found only in
simulations where the duration of spread was fixed
at 200 years (right contour graph), and where appro-
ximately 37% or more females survive to the mean
age at childbearing. In contrast, if the spread of the
LBK took place within 100 years (left contour graph)
it may be ruled out that it was the consequence only
of the migratory activity of farmers originating in
Transdanubia, because all TFR estimates at the 100%
density level are greater than the critical value of
fertility. The reliability of results of any demographic simula-
tion is directly dependent on the reliability of input
parameters. Thus, our motivation was to use only
sufficiently reliable parameters. Two of these (gene-
ration length and proportion of females at birth) are
assumed to be very stable across human populations
with natural reproduction (Hammel 1996). There
is no reason to speculate that they attained different
values in the population of LBK farmers. The dura-
tion of the spread of LBK has been estimated by nu-
merous independent analyses of radiocarbon data. There is a general agreement among scholars that
LBK spread from Transdanubia to the Rhine River
during 100–200 years. To achieve satisfactory con-
fidence of the survival of females, we collected a
large numbers of estimates (1000) gathered from
model life tables with widely ranging mortality lev-
els. On the other hand, the size of the origin and set-
tled area respectively we consider to be the input
parameters most prone to bias. Fortunately, the ana-
lysis of the effect of the input parameters has revea-
led (Tab. 3) that the sizes of the origin and settled
area have relatively low impact on the results of si-
mulations. Another important interpretation may be derived
from Figure 7. Discussion If we want to obtain acceptable esti-
mates of TFR (white parts of contour graphs), we
would have to assume that the population density of
farmers who spread from Transdanubia decreased
during the transition. To maintain the overall popu-
lation density in the settled area at 100%, a contri-
bution from local foragers to the establishment of
farming communities would have been necessary. What the admixture proportion was is a matter of
debate. If we assume the modal level of female sur-
vival (around 33%), then the proportion might be
10–30% of farmers to 90–70% of foragers if LBK ex-
panded during 100 years, or 10–50% of farmers to
90–50% of foragers if LBK expanded during 200
years. Such values of admixture proportion corre-
spond well to the results of genetic analyses that
have also implied a minor overall contribution from
Transdanubian farmers. Studies based on mtDNA
have suggested that the contribution of farmers was
between 13–20% (Richards and Macaulay 2000). According to Y-chromosome evidence, the genetic
contribution of Neolithic people may be as low as
22% (Semino et al. 2000). Although the majority of simulations gave unreal-
istically high estimates of TFR for the LBK popula-
tion, approximately 8% of them were concordant
with the hypothesis of colonization. The demogra-
phic conditions of colonization can be inferred from
the contour graphs in Figure 7. First, we suppose that
population density was maintained at a constant le-
vel during the expansion. That is to say, that the po-
pulation density after LBK expansion was as high as
the density in the origin area in Transdanubia. Pre-
viously, this assumption would have seemed unli-
kely, because population pressure was traditionally
viewed as the main trigger for the spread of the
Neolithic (Childe 1925). However, recent authors
agree that there is no solid evidence for population
pressure in Transdanubia that would encourage the
first farmers to migrate (Willis et al. 1998; Pavúk
2004), and that even Transdanubia was sparsely po-
pulated by people of the Earliest LBK (Whittle 1996). In our model, it is a priori assumed that the age and
sex structure of both admixing populations (immi-
grating foragers and expanding farmers) was identi-
cal. However, from the purely demographic view, a
fertility level is dependent only on the proportion of
females, not males. Discussion Van Bakel (1981) has given a growth
rate of 0.4 to 0.7% per annum for the period of Neo-
lithization, and similar values have been suggested
by Polgar (1972). Bandy (2001) has calculated that
the Neolithic population of the Basin of Mexico in
the Formative period grew at approximately 0.74%
per annum, and assumes that such a value is a very
high rate for an agricultural population with no ac-
cess to antibiotics or modern medicine. Neustupný
(1983) have assumed that a growth rate greater than
1% per annum for the Earliest LBK is highly unlike- Moreover, in our demographic projection, we as-
sume that LBK population enjoyed the most favor-
able conditions for population growth, because the
exponential function (Fig. 2) describes growth that
is unbounded by any factor. However, under more
realistic conditions, population growth is limited by
the carrying capacity of the environment, and the
growth rate gradually decreases to zero. Further-
more, we have presumed that stable and maximum
rate of growth was maintained during the entire
transition period and in the entire area settled at the
time. However, several authors argue that popula- Fig. 7. Contour graphs of the relationship among TFR (displayed as isolines), density ratio, and survival
of females for two temporal scenarios of the Earliest LBK expansion. For an explanation of the figures,
see text. Fig. 7. Contour graphs of the relationship among TFR (displayed as isolines), density ratio, and survival
of females for two temporal scenarios of the Earliest LBK expansion. For an explanation of the figures,
see text. 145 Patrik Galeta, Jaroslav Bruzek tion increase occurred only at the wave front, i.e. in
the relatively small contact zone between the expan-
ding farmers and indigenous foragers. In the area re-
maining, which is located behind the front, popula-
tion growth slows down (van Andel and Runnels
1995; Pinhasi 2003). Therefore, the actual level of
LBK fertility would have to be greater than we esti-
mated in the simulations. Thus, the colonization hy-
pothesis may be rejected with greater confidence. tion increase occurred only at the wave front, i.e. in
the relatively small contact zone between the expan-
ding farmers and indigenous foragers. In the area re-
maining, which is located behind the front, popula-
tion growth slows down (van Andel and Runnels
1995; Pinhasi 2003). ACKNOWLEDGEMENTS We would like to thank to Eszter Bánffy, Ivan Pavlů,
Vladimír Sládek, and Daniel Sosna for helpful com-
ments. Thanks to Daniel Sosna for English proofs of
the manuscript. All errors and omissions remain our
own. This research was supported by Junior grant KJB
708060701 of the Grant Agency of the Academy of
Sciences of the Czech Republic. ACKNOWLEDGEMENTS We would like to thank to Eszter Bánffy, Ivan Pavlů,
Vladimír Sládek, and Daniel Sosna for helpful com-
ments. Thanks to Daniel Sosna for English proofs of
the manuscript. All errors and omissions remain our
own. Conclusion In this paper we try to show that demographic sim-
ulations might be another independent line of evi-
dence in the study of the spread of agriculture in
Central Europe. We have demonstrated that the This research was supported by Junior grant KJB
708060701 of the Grant Agency of the Academy of
Sciences of the Czech Republic. REFERENCES Discussion To keep the overall fertility le-
vel of LBK population below the critical value of 6.92
children, immigration from forager communities
could have been sex-specific and limited only to fe-
males. This consequence inferred from the demogra-
phic model is well supported by other evidence. Ben-
tley (2007), based on strontium isotope analysis of
tooth enamel, has shown that female skeletons were 146 Demographic model of the Neolithic transition in Central Europe hypothesis of colonization proposed as the mecha-
nism of Neolithic transition in Central Europe may
be rejected in 92% of simulations. Colonization would
have been possible only if (1) the LBK population
was growing in the whole area throughout the tran-
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(3) the transition lasted at least 200 years. We have
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English
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Erratum to: Age and Tectonic Setting of the Kopri-Type Granitoids at the Junction Zone of the Dzhugdzhur–Stanovoi and Western Stanovoi Superterranes of the Central Asian Fold Belt
|
Doklady earth sciences
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a Institute of Precambrian Geology and Geochronology,
Russian Academy of Sciences, St. Petersburg, 199034 Russia
b Institute of the Earth’s Crust, Siberian Branch,
Russian Academy of Sciences, Irkutsk, 664033 Russia
*e-mail: larin7250@mail.ru ISSN 1028-334X, Doklady Earth Sciences, 2023, Vol. 513, Part 1, p. 1258. © The Author(s), 2023. This article is an open access publication. ISSN 1028-334X, Doklady Earth Sciences, 2023, Vol. 513, Part 1, p. 1258. © The Author(s), 2023. This article is an open access publication. Erratum to: Age and Tectonic Setting of the Kopri-Type Granitoids
at the Junction Zone of the Dzhugdzhur–Stanovoi
and Western Stanovoi Superterranes of the Central
Asian Fold Belt A. M. Larina,*, Corresponding Member of the RAS A. B. Kotova, E. B. Salnikovaa, S. D. Velikoslavinskiia,
V. P. Kovacha, T. M. Skovitinab, A. A. Ivanovaa, Yu. V. Plotkinaa, and N. Yu. Zagornayaa
Received November 16, 2023; revised November 16, 2023; accepted November 16, 2023 OPEN ACCESS The article “Age and Tectonic Setting of the Kopri-
Type Granitoids at the Junction Zone of the Dzhug-
dzhur–Stanovoi and Western Stanovoi Superterranes of
the Central Asian Fold Belt,” written by A.M. Larin,
A.B. Kotov, E.B. Salnikova, S.D. Velikoslavinskii,
V.P. Kovach, T.M. Skovitina, A.A. Ivanova, Yu.V. Plot-
kina, and N.Yu. Zagornaya, was originally published
electronically in Springer-Link on May 16, 2023 with-
out Open Access. After publication in volume 509,
Part 1, pages 111–117, the authors decided to make the
article an Open Access publication. Therefore, the copy-
right of the article has been changed to © The Author(s)
2023 and the article is forthwith distributed under the
terms of a Creative Commons Attribution 4.0 Interna-
tional License (http://creativecommons.org/licenses/
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https://doi.org/10.1134/S1028334X22601821 1258
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de
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XIII. Über die Natur „flüssiger“ und „fließender“ Krystalle
|
Zeitschrift für Kristallographie. Crystalline materials
| 1,909
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public-domain
| 2,328
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XIII. Über die Natur „flüssiger" und „fließender"
Krystalle.
Von
G. Wulff in Moskau.
Wenn man ein kleines Häufchen Kryställchen von P a r a a z o x y p h e n e t o l auf dem Objectgläschen schmilzt, mit einem ebenen Deckgläschen
bedeckt und abkühlt, so erstarrt bekanntlich die ganze Masse als ein
Aggregat nadeiförmiger, fächerartig gelagerter Krystalle von gelber Farbe.
Bringt man ein solches Präparat auf das Krystallisationsmikroskop und
erwärmt, so verwandelt es sich, nach O . L e h m a n n , in ein Aggregat
flüssiger Krystalle, die aber ihre Tropfenform wegen des Anhaftens an das
Glas nicht annehmen können und die Form der früheren festen Krystalle
nachahmen. Daß die doppeltbrechende Substanz am Glase haftet, davon
überzeugt man sich, wie 0. L e h m a n n gezeigt hat, dadurch, daß bei der
Verschiebung des Deckgläschens die obere Schicht mitgeführt wird und
die Contouren der doppeltbrechenden Felder sich in der Richtung der Verschiebung verdoppeln. Wenn man aber die Erscheinung näher beobachtet,
so kommt man zu der Überzeugung, daß z w i s c h e n d e n z w e i S c h i c h t e n
d o p p e l t b r e c h e n d e r S u b s t a n z sich eine i s o t r o p e F l ü s s i g k e i t bef i n d e t , denn bei der Verschiebung des Deckgläschens geht alles so vor
sich, als ob man zwei identische petrographische Präparate übereinander
verschöbe: es lassen sich keine störenden Nebenerscheinungen beobachten,
die die doppeltbrechende Wirkung der dazwischen liegenden Flüssigkeit an
den Tag brächten. Das ganze Präparat ist eine Schicht einfachbrechender
Flüssigkeit, die von unten und von oben von einem Häutchen doppeltbrechender, am Glase haftender Substanz umgeben ist. Am Rande des
Präparates, wo das Deckgläschen weggeschoben ist, kann man dieses Häutchen am Objectglase näher beobachten und es mit der Präpariernadel
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262
G. Wiilir.
kratzen.
An
solchen
Stellen beobachtet
man
auch
diejenigen
heftigen
Circulationen, die f ü r den unbedeckten Tropfen so charakteristisch sind.
Wenn man ein Häufchen Krystalle von Paraazoxyphenetol auf einem
Objectgläschen am Krystallisationsmikroskope bis zum Schmelzen
so beginnt es am Rande zu schmelzen, und in
stehen sofort heftige Strömungen.
einem
flüssigen
erwärmt,
dem Schmelzflusse ent-
Allmählich schmelzen alle Krystalle zu
T r o p f e n , auf dessen Scheitel
ein rundliches
flaches
Häut-
chen einer durchsichtigen Substanz schwimmt, deren Contouren sich fortwährend ändern.
Dieses Häutchen verschiebt sich hin und her unter der
Wirkung heftiger Stiümungen, die hauptsächlich an seinem Rande erzeugt
werden.
Am Häutchen haften winzige fremde Kürperchen, die als schwarze
Punkte erscheinen,
gegenseitige
Lage
sich mit dem Häutchen verschieben
nicht
ändern,
woraus
man
und dabei
ihre
muß,
daß
das
eine rasche
und
ge-
schließen
Häutchen nicht flüssig ist.
Man kann
das Häutchen
v o m Tropfen
durch
schickte B e w e g u n g der Präpariernadel wegschieben 1 ).
Mit dem Iläutchen
wird auch eine kleine Menge Flüssigkeit mitgerissen, und aus diesem Gemische bildet sich auf dem Objectgläschen ein Tropfen, dessen innere B e wegungen schon sehr langsam
und nur am Rande
merkbar
sind.
Aus
dieser Tatsache geht klar hervor, daß die Ursache der Strömungen in der
nicht homogenen Natur des Tropfens liegt,
aus
einer
aus
dieser Flüssigkeit
sich
der aus einer Flüssigkeit und
ausscheidenden
Substanz
besteht.
Beim Erkalten erstarrt dieser secundäre Tropfen, ganz wie der
primäre,
plötzlich zu einem fächerförmigen Aggregat nadeiförmiger Krystalle.
Beim
E r w ä r m e n werden im secundären Tropfen dieselben Erscheinungen beobachtet, wie in dem primären.
Nach
der Beseitigung
des Häutchens
der Circulationen viel deutlicher.
wird
der
gesamte
Charakter
Der ganze Tropfen befindet sich wie im
Sieden, seine Oberfläche teilt sich in
polygonale,
Felder,
heftiger innerer Circulationen
fort-
eben
könnte
man
der Ursache
der
deren Contouren aber wegen
während
wechseln.
glauben,
daß mit der Beseitigung
Strömungen,
Nach dem,
was
gesagt
des H ä n i c h e n s ,
die letzteren a u f h ö r e n sollten.
gewöhnlich
wurde,
als
E s erübrigt
fünfeckige
sich aber sehr
leicht, daß das Häutchen nicht die ganze aus des Flüssigkeit abgeschiedene
Menge doppeltbrechender Substanz darstellt: der Boden der Tropfens zeigt
doppeltbrechende Felder.
Wenn man diesen B o d e n ,
d. h. die Oberfläche
des Objectgläschens mit dem Stereomikroskope beobachtet, so merkt man
sofort, daß diese Oberfläche mit einem unebenen Häutchen überzogen ist,
von dessen Punkten die Strömungen
ausgehen.
1 ) Die E i n f ü h r u n g d e r S p i t z e einer P r ä p a r i e r n a d e l e r h ö h t b e d e u t e n d die E n e r g i e
der
Circulation.
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Über die Natur »flüssiger« und f l i e ß e n d e r «
Krystalle.
263
W a s die Girculationen anbetrifft, so sind sie sehr gut im gewöhnlichen,
unpolarisierten Lichte sichtbar, sehr scharfe Schlieren bildend. Die Girculationen bestehen also in Concentrationsströmungen.
E r w ä r m t m a n den Tropfen w e i t e r , bis zur Klärungstemperatur, so
bemerkt m a n , d a ß die Klärung vom Rande des Tropfens aus beginnt.
Das doppeltbrechende Feld zieht sich an dem Scheitel des Tropfens zusammen und wird von einem Kränzchen einzelner doppeltbrechender
Tröpfchen begleitet, die sich r a s c h bilden, zusammenfließen und verschwinden. Diese Tröpfchen sind den »flüssigen Krystallen« von P a r a a z o x y phenetol vollständig ähnlich. Im Inneren dieser Tröpfchen beobachtet man
eben solche Girculationen, wie in dem ganzen Tropfen. Beim Zusammenziehen des doppeltbrechenden Feldes bleiben die Girculationen nur in demselben und in den das Feld umgebenden Tröpfchen übrig: die sich klärende
Flüssigkeit wird ganz ruhig.
Beim Klärungspunkte verschwinden die
Tröpfchen am Scheitel des Tropfens. Wenn das Häutchen nicht weggenommen w u r d e , so verschwindet es gleichzeitig und es bleiben n u r die
fremden Körperchen übrig, die ein zusammenhängendes Netzchen bilden
und auf der Oberfläche des Tropfens sich hin und her verschieben. Wenn
man jetzt die im Inneren des Tropfens schwebenden fremden Körperchen
beobachtet, so bemerkt man zwar die Conveclionsströmungen, doch sind
diese Strömungen sehr undeutlich und langsam im Vergleich mit den
früheren Girculationen und bilden keine Schlieren.
L ä ß t man den isotrop gewordenen Tropfen sich a b k ü h l e n , so erscheinen plötzlich kleine gelbe doppeltbrechende Tröpfchen an den auf der
Oberfläche des Tropfens schwimmenden f r e m d e n Körperchen und fließen
rasch zu einem Häutchen zusammen. Um das Häutchen bildet sich ein
Kränzchen aus solchen Tröpfchen, die mit einander und mit der Subslanz
des Häutchens zusammenfließen.
Es entstehen die Circulalionen in dem
sich ausbreitenden doppeltbrechenden Felde, das sich endlich bis zum
Rande des Tropfens ausdehnt. Die Erscheinung behält im Ganzen ihren
Charakter w ä h r e n d der Abkühlung bis zum plötzlichen Erscheinen der
festen nadeiförmigen Krystalle.
W e n n man beim E r w ä r m e n das Häutchen beseitigt h a t , so ist das
Erscheinen der gelben Tröpfchen bei der Abkühlung der isotropen Flüssigkeit sehr auffallend. Es entstehen winzige T r ö p f c h e n , die ein schwarzes,
oft spiralförmig gewundenes Kreuzchen zwischen gekreuzten Nicols sehr
deutlich zeigen. Die Tröpfchen lagern sich nach den zerrissenen Seiten
der Fünfecke, so d a ß die größeren in den Eckpunkten, die kleineren längs
der Seiten sich ordnen. Alles befindet sich in heftiger Bewegung, die
Größe und die gegenseitige Lage der Tröpfchen ä n d e r t sich fortwährend,
die Tröpfchen verschwinden und erscheinen wieder. Mit sinkender Tem-
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264
G. W u l f f .
p e r a t u r nehmen die Tröpfchen an Größe zu u n d m a n sieht ganz deutlich, daß ihr Inneres sich in heftiger Circulation befindet. Die Tröpfchen
fließen zu den fünfeckigen Feldern z u s a m m e n , innerhalb deren die Cireulationen sich so ordnen, d a ß in der Mitte des Feldes der Strom steigt, am
Rande aber sinkt. Die ganze Erscheinung breitet sich bis zum Rande des
Tropfens aus, wie es schon oben beschrieben wurde.
Nach der Beseitigung des Häutchens ist die Menge der sich aus der
Flüssigkeit abscheidenden Substanz offenbar kleiner.
Im innigen Zusammenhange mit dieser Tatsache steht die folgende, die sich nach Beseitigung
des Häutchens beobachten läßt und die auf die ganze Natur der »flüssigen
Krystalle« von P a r a a z o x y p h e n e t o l ein klares Licht w i r f t : d i e g r o ß e n ,
g e g e n den R a n d des T r o p f e n s sich b i l d e n d e n T r ö p f c h e n zeigen
o f t I n t e r f e r e n z f ä r b e n . Diese Tröpfchen sind also optisch d ü n n e r , als
die kleineren, f r ü h e r sich bildenden Tröpfchen. Diese p a r a d o x e Tatsache
kann meiner Meinung nach n u r eine einzige mögliche Erklärung h a b e n :
die T r ö p f c h e n s i n d mit i s o t r o p e r F l ü s s i g k e i t g e f ü l l t e B l a s e n ,
deren Hülle d o p p e l t b r e c h e n d ist.
Wenn es an der die Hülle bildenden Substanz mangelt, so wird die Hülle dünner und es erscheinen die
Interferenzfarben niedriger Ordnung. Nun können aber die gelben T r ö p f chen nichts a n d e r e s , als die »flüssigen Krystalle« von P a r a a z o x y p h e n e t o l
sein, und wenn diese Tröpfchen Blasen sind, so sind es auch diese Krystalle.
Wir kommen also zu dem Schlüsse, d a ß d i e » f l ü s s i g e n K r y s t a l l e «
von P a r a a z o x y p h e n e t o l S c h a u m z e l l e n sind.
Jetzt werden auch die Erscheinungen verständlich, die beim Verschieben des Deckgläschens sich beobachten lassen — die Erscheinung der
oberen und unteren am Glase haftenden Häutchen und die Circulationen
am Rande.
Es entsteht jetzt eine ganze Reihe von Fragen: Was ist die Substanz
des Häutchens, die die Hüllen der Blasen bildet, in welcher Weise e n t s t e h t
sie und ist ihre Menge constant, oder wird sie bei der beireffenden T e m p e r a t u r immer gebildet? W o h e r r ü h r t die Doppelbrechung der Blasenhülle
— sind es Spannungen, die diese Doppelbrechung h e r v o r r u f e n ? Alle diese
Fragen bedürfen specieller Untersuchungen.
Wenn die Natur der »flüssigen Krystalle« von P a r a a z o x y p h e n t o l sich
sehr schwierig erkennen l ä ß t , so ist diese Aufgabe f ü r »fließende Krystalle« von P a r a a z o x y b e n z o e s ä u r e ä t h y l e s t e r viel einfacher; man
braucht nur das Stereomikroskop zu Hilfe zu nehmen. Man sieht dann
sehr deutlich, daß diese »fließenden Krystalle« aus kleinen pleochro'itischen
ovalen, zugespitzten, blätterförmigen Kryställchen bestehen, die in eine
flüssige Hülle eingebettet sind. Diese Hülle wirkt capillar auf die Anordnung der von ihr umhüllten Kryställchen. Wenn das P r ä p a r a t unbedeckt
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Über d i e N a t u r
»flüssiger«
und
ist, so ist die Anordnung sphärolitisch,
»fließender«
Kryslalle.
entsprechend
265
der kugeligen F o r m
der flüssigen Hülle; wenn das Präparat aber mit dem Deckgläschen bedeckt
ist, so ist die Hülle flach und dementsprechend ordnen sich die Kryställchen in einer E b e n e ,
wo sie mit einander entweder parallel oder in der
Zwillingsstellung sich verbinden.
Wenn
man dieses Ordnen
der Kryställ-
chen von Paraazoxybenzoesäureäthylester
nach den beiden
obigen Lagen
beobachtet, die Lagen, die durch scharfe Bewegungen der Kryställchen erzielt werden, so entsteht der Gedanke, ob nicht überhaupt die Zwillinge
ihre Entstehung äußeren capillaren Kräften verdanken.
Als ich nach den oben beschriebenen Beobachtungen den prachtvollen
Atlas
des L e h m a n n s c h e n
Werkes
»Flüssige
Kryslalle,
sowie
Plasticität
usw.« nochmals durchblätterte, fiel mir ins Auge, daß die Contouren der
flüssigen Krystalle sehr oft doppelt erscheinen (s. besonders Taf. X, 2 und
Taf. XXIV, 3), was keineswegs durch optische Täuschung erklärt werden
kann und gerade auf eine zellenartige Natur dieser »Krystalle« hinweist.
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https://openalex.org/W3125427995
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http://repositorio.lneg.pt/bitstream/10400.9/3217/1/Molecules_Vol.24_article808.pdf
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English
| null |
The Effect of Chemical Character of Ionic Liquids on Biomass Pre-Treatment and Posterior Enzymatic Hydrolysis
| null | 2,019
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cc-by
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Joana R. Bernardo, Francisco M. Gírio and Rafał M. Łukasik * Joana R. Bernardo, Francisco M. Gírio and Rafał M. Łukasik * Joana R. Bernardo, Francisco M. Gírio and Rafał M. Łukasik
Unidade de Bioenergia, Laboratório Nacional de Energia e Geologia, I.P., Estrada do Paço do Lumiar 22,
1649-038 Lisboa, Portugal; joana.bernardo@lneg.pt (J.R.B.); francisco.girio@lneg.pt (F.M.G.)
* Correspondence: rafal.lukasik@lneg.pt
Academic Editors:
Ivet Ferrer, Cigdem Eskicioglu, Georgia Antonopoulou and
Audrey Battimelli
Received: 3 February 2019; Accepted: 20 February 2019; Published: 23 February 2019
Joana R. Bernardo, Francisco M. Gírio and Rafał M. Łukasik
Unidade de Bioenergia, Laboratório Nacional de Energia e Geologia, I.P., Estrada do Paço do Lumiar 22,
1649-038 Lisboa, Portugal; joana.bernardo@lneg.pt (J.R.B.); francisco.girio@lneg.pt (F.M.G.)
* Correspondence: rafal.lukasik@lneg.pt
Academic Editors:
Ivet Ferrer, Cigdem Eskicioglu, Georgia Antonopoulou and
Audrey Battimelli
R
i
d 3 F b
2019 A
t d 20 F b
2019 P bli h d 23 F b
2019
Unidade de Bioenergia, Laboratório Nacional de Energia e Geologia, I.P., Estrada do Paço do Lumiar 2
1649-038 Lisboa, Portugal; joana.bernardo@lneg.pt (J.R.B.); francisco.girio@lneg.pt (F.M.G.)
* Correspondence: rafal.lukasik@lneg.pt Academic Editors:
Ivet Ferrer, Cigdem Eskicioglu, Georgia Antonopoulou and
Audrey Battimelli
Received: 3 February 2019; Accepted: 20 February 2019; Published: 23 February 2019 Abstract: Ionic liquids have been recognised as interesting solvents applicable in efficient lignocellulosic
biomass valorisation, especially in biomass fractionation into individual polymeric components or
direct hydrolysis of some biomass fractions. Considering the chemical character of ionic liquids, two
different approaches paved the way for the fractionation of biomass. The first strategy integrated
a pre-treatment, hydrolysis and conversion of biomass through the employment of hydrogen-bond
acidic 1-ethyl-3-methyimidazolim hydrogen sulphate ionic liquid. The second strategy relied on
the use of a three-step fractionation process with hydrogen-bond basic 1-ethyl-3-methylimidazolium
acetate to produce high purity cellulose, hemicellulose and lignin fractions. The proposed approaches
were scrutinised for wheat straw and eucalyptus residues. These different biomasses enabled an
understanding that enzymatic hydrolysis yields are dependent on the crystallinity of the pre-treated
biomass. The use of acetate based ionic liquid allowed crystalline cellulose I to change to cellulose II and
consequently enhanced the glucan to glucose yield to 93.1 ± 4.1 mol% and 82.9 ± 1.2 mol% for wheat
straw and eucalyptus, respectively. However, for hydrogen sulphate ionic liquid, the same enzymatic
hydrolysis yields were 61.6 ± 0.2 mol% for wheat straw and only 7.9 ± 0.3 mol% for eucalyptus residues. Joana R. Bernardo, Francisco M. Gírio and Rafał M. Łukasik * These results demonstrate the importance of both ionic liquid character and biomass type for efficient
biomass processing. ywords: biomass; valorisation; ionic liquid; crystallinity; enzymatic hydrolysis; pre-treatment Keywords: biomass; valorisation; ionic liquid; crystallinity; enzymatic hydrolysis; pre-treatment molecules molecules Molecules 2019, 24, 808; doi:10.3390/molecules24040808 www.mdpi.com/journal/molecules molecules molecules 1. Introduction Lignocellulosic biomass is a renewable, sustainable, abundant, and CO2 neutral alternative to
fossil feedstock for a portfolio of fuels, chemicals and materials. Composed of crystalline cellulose
nanofibrils embedded in an amorphous matrix of cross-linked lignin and hemicelluloses, lignocellulose
shows a natural recalcitrance that impedes enzyme and microbial accessibility, resulting in the relatively
low digestibility of raw lignocellulosic materials [1]. Thus, an efficient pre-treatment, and consequently
a deconstruction of the lignocellulosic biomass, makes these fractions susceptible for more favourable
transformation to value-added products [2,3]. However, many pre-treatment methods require harsh
conditions, especially temperature and/or pressure that often result in undesired by-products, which
decrease the sugar yields and inhibit enzymatic hydrolysis and further bioconversion [2]. In recent years, ionic liquids (ILs) have gained increasing interest for biomass processing due to
their capacity to dissolve lignocellulosic biomass by an effective disruption of the complex network
of noncovalent interactions between carbohydrates and lignin [4–6]. A main function of IL in
lignocellulosic biomass pre-treatment is the modification the fibrillary structure of cell walls in order Molecules 2019, 24, 808; doi:10.3390/molecules24040808 www.mdpi.com/journal/molecules 2 of 17 Molecules 2019, 24, 808 to: (i) decrease cellulose crystallinity, (ii) increase cellulose surface accessibility by the removal of lignin
and/or hemicellulose, and (iii) promotion of a swelling effect of the biomass [7]. Imidazolium-based
ILs are among the most extensively studied ILs and have demonstrated that either a cation or an
anion is of considerable importance in biomass processing [8,9]. Swatloski et al. showed that a
high concentration of chloride anions is effective in breaking down the hydrogen-bond network of
cellulose [10]. A similar effect was reported for the acetate ([OAc]) anion, which was demonstrated to
be efficient in the dissolution of lignocellulosic biomass [11]. It was reported that a key reason for this
was the high hydrogen bond acceptor capacity (β) of the [OAc] anion (β = 1.201) in comparison to
previously mentioned chloride anion (β = 0.83) [12]. Due to this, 1-ethyl-3-methylimidazolium acetate
IL is confirmed to be one of the best and is one of the most commonly used [13] ILs, able to dissolve
a large variety of lignocellulosic biomass and to fractionate it into cellulose- and hemicellulose-rich
fractions, as well as to produce high pure lignin [9,14,15]. An alternative approach to biomass processing with ILs is the employment of ILs as both solvent
and catalyst. 1. Introduction In these processes, ILs hydrolyse mainly polysaccharide fractions without the presence
of other catalysts [5,16,17]. Therefore, ILs with an acidic character have demonstrated an ability to
selectively hydrolyse hemicellulose [18,19], both cellulose and hemicellulose [20], or lignin [21]. Some
of the most commonly used ILs in such approaches are those based on hydrogen sulphate ([HSO4]). They are able to catalyse a selective hemicellulose hydrolysis [17–19]. Furthermore, [HSO4]-based
ILs have been increasingly used because of their acidic properties and due to their low cost when
compared to other ILs [22]. The use of acidic IL, e.g., 1-butyl-3-methylimidazolium hydrogen sulphate,
[bmim][HSO4], allowed achievement of up to 90% fermentable glucose after enzymatic saccharification
of the cellulose-rich Miscanthus pulp [18]. The same IL was also reported as being able to hydrolyse
and to convert the hemicellulose fraction of wheat straw with no additional catalyst used [17]. The pre-treatment with this IL produced a liquor enriched in hemicellulosic sugars, furans and
organic acids, and a solid fraction constituted mainly of cellulose and lignin. Furthermore, water was
confirmed to have an influence on the equilibrium of the hemicellulose hydrolysis. The increase of the
water content close to 10% (w/w) in the reaction system disfavoured xylose dehydration, and thus
allowed the production of hemicellulose-derived monosaccharides to increase significantly [19]. Building on previous works about the processing of biomass with hydrogen-bond basic
([emim][OAc]) [11] and hydrogen-bond acidic ([emim][HSO4]) [17,19] ILs, this work aimed to
demonstrate the importance of biomass type on the efficiency of the biomass pre-treatment, as well
as on the efficiency of subsequent enzymatic hydrolysis. This was examined using two very distinct
types of biomasses, namely, herbaceous (wheat straw) and hardwood Eucalyptus globulus, allowing the
elucidation of changes observed in the chemical structure of the biomass, cellulose crystallinity and
consequently the effects of these on the cellulose-rich pulp hydrolysability. 2. Results and Discussion 2. Results and Discussion 2.1. Hydrogen-Bond Acidic IL 2.1.1. Biomass Pre-Treatment with [emim][HSO4] The first methodology used focused on the biomass pre-treatment with [emim][HSO4]. As stated
above, this approach allowed the integration of biomass pre-treatment, hydrolysis and conversion in a
single-step process. The acidic character of the [HSO4] anion of IL, promoted a selective hydrolysis of
the hemicellulose fraction, and the resulting products (mainly pentoses and furfural) were kept in the
liquid phase. Both biomasses, wheat straw and eucalyptus residues, were subject to processing with
[emim][HSO4] IL at 140 ◦C for 90 min at varied [emim][HSO4]/H2O mass ratio and with fixed 10 wt.%
of dry biomass in the reaction mixture. The pentose and furfural yields obtained in these trials are depicted in Figure 1. 3 of 17
3 of 17 Molecules 2019, 24, 808
Molecules 2019, 24, x Figure 1. The yields of (●○) pentose (sum of arabinose and xylose) or (■□) furfural as a function of
ionic liquid (IL) concentration (wt.%) obtained from pre-treatment of wheat straw (filled symbols)
and eucalyptus residues (open symbols) performed at 140 °C for 90 min. IL (wt.%)
20
30
40
50
60
Pentose or furfural yield (mol%)
0
20
40
60
80
Figure 1. The yields of (•#) pentose (sum of arabinose and xylose) or (■□) furfural as a function of
ionic liquid (IL) concentration (wt.%) obtained from pre-treatment of wheat straw (filled symbols) and
eucalyptus residues (open symbols) performed at 140 ◦C for 90 min. Figure 1. The yields of (●○) pentose (sum of arabinose and xylose) or (■□) furfural as a function of
ionic liquid (IL) concentration (wt.%) obtained from pre-treatment of wheat straw (filled symbols)
IL (wt.%)
20
30
40
50
60
Pentose or furfural yield (mol%)
0
20
40
60
80
igure 1. The yields of (•#) pentose (sum of arabinose and xylose) or (■□) furfural as a function of
onic liquid (IL) concentration (wt.%) obtained from pre-treatment of wheat straw (filled symbols) and Figure 1. The yields of (●○) pentose (sum of arabinose and xylose) or (■□) furfural as a function of
ionic liquid (IL) concentration (wt.%) obtained from pre-treatment of wheat straw (filled symbols)
and eucalyptus residues (open symbols) performed at 140 °C for 90 min. Figure 1. The yields of (•#) pentose (sum of arabinose and xylose) or (■□) furfural as a function of
ionic liquid (IL) concentration (wt.%) obtained from pre-treatment of wheat straw (filled symbols) and
eucalyptus residues (open symbols) performed at 140 ◦C for 90 min. 2.1.1. Biomass Pre-Treatment with [emim][HSO4] As can be seen, at IL concentration of 30 wt.%, the pentose yields peaked for both biomasses. For
eucalyptus residues or wheat straw, a further increase in the IL concentration was demonstrated to
have a negative effect on the pentose yield as it was counterbalanced by predominant production of
furfural. These results are not surprising since a high performance of acidic ILs towards furfural is
often reported in the literature [23–25]. One of the reasons for this is that pentose conversion to
furfural is a dehydration reaction; therefore, a higher concentration of IL, or in other words a lower
concentration of water, may disturb the equilibrium existing in the system promoting the
dehydration of pentoses towards furfural production. On the other hand, the presence of water in
the system allowed protection of pentoses from dehydration, and consequently, it was possible to
obtain a high pentose yield for IL concentration below 30 wt.%. Although this observation is valid
for either wheat straw or eucalyptus residues, the yield of hemicellulose hydrolysis, as well as
pentose and furfural yields, seems to be strongly dependent on the nature of the biomass. As can be
seen in Figure 1, in the best conditions, i.e., 30 wt.% IL, pre-treatment of the eucalyptus residues with
[emim][HSO4] allowed achievement of only 37.9 ± 1.7 mol% pentoses, while for wheat straw the
maximum pentose concentration was almost double, i.e., 70.1 ± 0.5 mol%. This difference is also
reflected in the composition of pre-treated solids and the solid yields. The latter was very high for
eucalyptus residues and varied between 81 and 85 wt.%, while for pre-treated wheat straw it
oscillated between 68 and 75 wt.%. For both biomasses, the lowest solid yields were observed for
solids obtained following pre-treatment with an IL concentration of 30 wt.%. This is a direct
consequence of the most pronounced hydrolysis of hemicellulose. Further increase in the IL
concentration increased the solid yield for wheat straw up to 74.9 ± 3.0 wt.%. This unexpected increase
of the solid yield with an increase in the pre-treatment intensity might be justified by the formation
of pseudo-lignins, also called humins [26]. As pseudo-lignins are quantified gravimetrically [27], they
contribute to an increase in the lignin content detected, which is clearly visible in Figure 2. For two
the highest IL concentrations tested, the lignins recovered exceeded 100 wt.% of the lignin present in
the native biomass. 2.1.1. Biomass Pre-Treatment with [emim][HSO4] This, in turn may confirm this hypothesis. As can be seen, at IL concentration of 30 wt.%, the pentose yields peaked for both biomasses. For eucalyptus residues or wheat straw, a further increase in the IL concentration was demonstrated
to have a negative effect on the pentose yield as it was counterbalanced by predominant production
of furfural. These results are not surprising since a high performance of acidic ILs towards furfural
is often reported in the literature [23–25]. One of the reasons for this is that pentose conversion
to furfural is a dehydration reaction; therefore, a higher concentration of IL, or in other words a
lower concentration of water, may disturb the equilibrium existing in the system promoting the
dehydration of pentoses towards furfural production. On the other hand, the presence of water in
the system allowed protection of pentoses from dehydration, and consequently, it was possible to
obtain a high pentose yield for IL concentration below 30 wt.%. Although this observation is valid for
either wheat straw or eucalyptus residues, the yield of hemicellulose hydrolysis, as well as pentose
and furfural yields, seems to be strongly dependent on the nature of the biomass. As can be seen
in Figure 1, in the best conditions, i.e., 30 wt.% IL, pre-treatment of the eucalyptus residues with
[emim][HSO4] allowed achievement of only 37.9 ± 1.7 mol% pentoses, while for wheat straw the
maximum pentose concentration was almost double, i.e., 70.1 ± 0.5 mol%. This difference is also
reflected in the composition of pre-treated solids and the solid yields. The latter was very high for
eucalyptus residues and varied between 81 and 85 wt.%, while for pre-treated wheat straw it oscillated
between 68 and 75 wt.%. For both biomasses, the lowest solid yields were observed for solids obtained
following pre-treatment with an IL concentration of 30 wt.%. This is a direct consequence of the most
pronounced hydrolysis of hemicellulose. Further increase in the IL concentration increased the solid
yield for wheat straw up to 74.9 ± 3.0 wt.%. This unexpected increase of the solid yield with an
increase in the pre-treatment intensity might be justified by the formation of pseudo-lignins, also called
humins [26]. As pseudo-lignins are quantified gravimetrically [27], they contribute to an increase in
the lignin content detected, which is clearly visible in Figure 2. 2.1.1. Biomass Pre-Treatment with [emim][HSO4] The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 °C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. Figure 2. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark
grey bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 ◦C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. Figure 2. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 °C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. Figure 2. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark
grey bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 ◦C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. Figure 3. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of eucalyptus residues pre-treatment at 140 °C and 90 min with
[emim][HSO4] at various % IL The solid line represents the solid yield (wt %) of pre-treated solids
0
20
40
60
80
100
0
20
40
60
80
100
Solid composition (wt%)
IL (wt.%)
native
20.0
30.0
41.3
50.0
Solid Yield (wt.%)
Figure 3. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of eucalyptus residues pre-treatment at 140 °C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. 0
20
40
60
80
100
0
20
40
60
80
100
Solid composition (wt%)
IL (wt.%)
native
20.0
30.0
41.3
50.0
Solid Yield (wt.%)
Figure 3. 2.1.1. Biomass Pre-Treatment with [emim][HSO4] The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of eucalyptus residues pre-treatment at 140 ◦C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. Figure 3. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of eucalyptus residues pre-treatment at 140 °C and 90 min with
[
i
][HSO ]
t
i
% IL Th
lid li
t th
lid
i ld (
t %)
f
t
t d
lid
IL (wt.%)
na
2
3
4
Figure 3. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of eucalyptus residues pre-treatment at 140 °C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. Figure 3. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of eucalyptus residues pre-treatment at 140 ◦C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. recovered after pre-treatment. The aforementioned analysis of the hemicellulose hydrolysis also reflects the macromolecular
composition of the pre-treated leftovers of both biomasses. It is clear that the IL pre-treatment
induced the reduction of the hemicellulose fraction in comparison to native biomasses, as xylan was
found in amounts not exceeding 15 wt.% of the pre-treated biomasses. Consequently, the major
components of the solids produced were glucan, followed by lignin, as already discussed. This
indicates that contrary to a great performance by aqueous [emim][HSO4] solution in processing
hemicellulose, a cellulose hydrolysis in this catalytic system seems to be very inefficient. For IL
concentrations up to the studied 41.3 wt.%, the cellulose yield was kept constant, but in case of wheat
straw, for higher IL concentrations (>50 wt.%), the cellulose content started to diminish. This
demonstrates that only under these conditions the cellulose fraction of wheat straw was also
The aforementioned analysis of the hemicellulose hydrolysis also reflects the macromolecular
composition of the pre-treated leftovers of both biomasses. It is clear that the IL pre-treatment
induced the reduction of the hemicellulose fraction in comparison to native biomasses, as xylan was
found in amounts not exceeding 15 wt.% of the pre-treated biomasses. 2.1.1. Biomass Pre-Treatment with [emim][HSO4] For two the highest IL concentrations
tested, the lignins recovered exceeded 100 wt.% of the lignin present in the native biomass. This,
in turn may confirm this hypothesis. ,
y
yp
Similar effects were not observed for eucalyptus residues, for which either solid yield appeared
to be constant or the lignin content in the solids pre-treated at various IL concentrations did not
change, as demonstrated in Figure 3. y
yp
Similar effects were not observed for eucalyptus residues, for which either solid yield appeared to
be constant or the lignin content in the solids pre-treated at various IL concentrations did not change,
as demonstrated in Figure 3. 4 of 17
4 of 17 Molecules 2019, 24, 808
Molecules 2019, 24, x Figure 2. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 °C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. 0
20
40
60
80
100
0
20
40
60
80
100
Solid composition (wt%)
IL (wt.%)
native
20.0
30.0
41.3
50.0
60.0
Solid Yield (wt.%)
Figure 2. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark
grey bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 ◦C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. Figure 2. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
bar–Klason lignin; black bar–others) of wheat straw pre-treatment at 140 °C and 90 min with
[emim][HSO4] at various % IL. The solid line represents the solid yield (wt.%) of pre-treated solids
recovered after pre-treatment. 0
20
40
60
80
0
20
40
60
80
Solid composition (wt%)
IL (wt.%)
native
20.0
30.0
41.3
50.0
60.0
Solid Yield (wt.%) 0
20
40
60
80
100
0
20
40
60
80
100
Solid composition (wt%)
IL (wt.%)
native
20.0
30.0
41.3
50.0
60.0
Solid Yield (wt.%)
Figure 2. The solid phase composition (white bar–cellulose; light grey bar–hemicellulose; dark grey
0
20
40
60
80
0
20
40
60
80
Solid composition (wt%)
IL (wt.%)
native
20.0
30.0
41.3
50.0
60.0
Solid Yield (wt.%) Figure 2. 2.1.1. Biomass Pre-Treatment with [emim][HSO4] They obtained total reducing sugars (TRS) yields of 23% and 15% from corn stalk after only
5 min of the reaction at 100 °C in [bmim][HSO4] and [C4SO3Hmim][HSO4], respectively. On the other
hand, longer reaction times provided lower TRS yields, suggesting that these strongly acidic ILs
resulted in the promotion of more advanced degradation of polysaccharide fraction at the pre-
treatment step [20]. Other work showed that in case of Miscanthus, pre-treatment at a higher
temperature (120 °C) and for 4 h with [bmim][HSO4] and 20 vol.% (17 wt.%) of H2O content, close to
16% of hemicellulose sugar monomers were obtained [18]. In a different study, Carvalho et al. showed that the same IL with 9.22 wt.% H2O content in the pre-treatment of wheat straw (125 °C and
82.1 min) allowed a 40.1 mol% pentose yield to be obtained [19]. Thus, comparing the data in the
literature to the data presented in this work, it can be stated that a higher yield of pentoses was
selectively achieved without excessive amounts of IL being present in the system. 2.1.2. Enzymatic Hydrolysis of Pre-Treated Solids 2.1.1. Biomass Pre-Treatment with [emim][HSO4] Consequently, the major
components of the solids produced were glucan, followed by lignin, as already discussed. This
indicates that contrary to a great performance by aqueous [emim][HSO4] solution in processing
hemicellulose, a cellulose hydrolysis in this catalytic system seems to be very inefficient. For IL
concentrations up to the studied 41.3 wt.%, the cellulose yield was kept constant, but in case of wheat
straw, for higher IL concentrations (>50 wt.%), the cellulose content started to diminish. This
demonstrates that, only under these conditions, the cellulose fraction of wheat straw was also
The aforementioned analysis of the hemicellulose hydrolysis also reflects the macromolecular
composition of the pre-treated leftovers of both biomasses. It is clear that the IL pre-treatment induced
the reduction of the hemicellulose fraction in comparison to native biomasses, as xylan was found in
amounts not exceeding 15 wt.% of the pre-treated biomasses. Consequently, the major components
of the solids produced were glucan, followed by lignin, as already discussed. This indicates that
contrary to a great performance by aqueous [emim][HSO4] solution in processing hemicellulose,
a cellulose hydrolysis in this catalytic system seems to be very inefficient. For IL concentrations up
to the studied 41.3 wt.%, the cellulose yield was kept constant, but in case of wheat straw, for higher
IL concentrations (>50 wt.%), the cellulose content started to diminish. This demonstrates that, only
under these conditions, the cellulose fraction of wheat straw was also susceptible to hydrolysis, and for
pre-treatment with IL concentration of 60 wt.% as much as 20 wt.% of cellulose, in comparison to native 5 of 17
20 wt.%
i
th Molecules 2019, 24, 808
susceptible to hyd biomass, was removed. In the case of eucalyptus residues, in the range of IL concentrations studied,
20 wt.% of IL already allowed the removal of about 1/3 of hemicellulose and further increases in the
IL concentration in the reaction mixture resulted in no further significant changes in the composition
of the pre-treated solids, as can be seen in Figure 3. These results confirm again that although ILs
are capable of processing various types of herbaceous and woody biomass as a single feedstock,
the conditions for efficient pre-treatment are dependent upon the biomass type, with softwoods or
hardwoods recognised as the most difficult to process when compared to herbaceous biomass [6]. Indeed, Xu et al. 2.1.1. Biomass Pre-Treatment with [emim][HSO4] found that the eucalyptus residues treatment catalysed by 0.5% (v·v−1) [bmim][HSO4]
yielded only ≈25 mol% of pentose at 190 ◦C [28]. Other reports in the literature depict the use of
the acidic ILs in the pre-treatment of biomass as well. For example, Li et al. demonstrated acidic IL
to be an efficient system for the hydrolysis of lignocellulosic materials. They obtained total reducing
sugars (TRS) yields of 23% and 15% from corn stalk after only 5 min of the reaction at 100 ◦C in
[bmim][HSO4] and [C4SO3Hmim][HSO4], respectively. On the other hand, longer reaction times
provided lower TRS yields, suggesting that these strongly acidic ILs resulted in the promotion of
more advanced degradation of polysaccharide fraction at the pre-treatment step [20]. Other work
showed that in case of Miscanthus, pre-treatment at a higher temperature (120 ◦C) and for 4 h with
[bmim][HSO4] and 20 vol.% (17 wt.%) of H2O content, close to 16% of hemicellulose sugar monomers
were obtained [18]. In a different study, Carvalho et al. showed that the same IL with 9.22 wt.% H2O
content in the pre-treatment of wheat straw (125 ◦C and 82.1 min) allowed a 40.1 mol% pentose yield
to be obtained [19]. Thus, comparing the data in the literature to the data presented in this work, it
can be stated that a higher yield of pentoses was selectively achieved without excessive amounts of IL
being present in the system. range of IL concentrations studied, 20 wt.% of IL already allowed the removal of about 1/3 of
hemicellulose and further increases in the IL concentration in the reaction mixture resulted in no
further significant changes in the composition of the pre-treated solids, as can be seen in Figure 3. These results confirm again that although ILs are capable of processing various types of herbaceous
and woody biomass as a single feedstock, the conditions for efficient pre-treatment are dependent
upon the biomass type, with softwoods or hardwoods recognised as the most difficult to process
when compared to herbaceous biomass [6]. Indeed, Xu et al. found that the eucalyptus residues
treatment catalysed by 0.5% (v⋅v−1) [bmim][HSO4] yielded only ≈ 25 mol% of pentose at 190 °C [28]. Other reports in the literature depict the use of the acidic ILs in the pre-treatment of biomass as well. For example, Li et al. demonstrated acidic IL to be an efficient system for the hydrolysis of lignocellulosic
materials. 2.1.2. Enzymatic Hydrolysis of Pre-Treated Solids
The efficiency of pre-treatment of wheat stra
l
d b
i
h d
l
i
Th The efficiency of pre-treatment of wheat straw and eucalyptus residues by [emim][HSO4] was
evaluated by enzymatic hydrolysis. The enzymatic hydrolysis yield of pre-treated wheat straw is
presented in Figure 4. evaluated by enzymatic hydrolysis. The enzymatic hydrolysis yield of pre-treated wheat straw is
presented in Figure 4. Figure 4. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of wheat straw pre-treated solids as a function of IL concentration used in the pre-treatment. The enzymatic hydrolysis yield for native wheat straw is presented for comparison. IL (wt.%)
Hydrolysis yield (mol%)
0
20
40
60
80
native
20.0
30.0
41.3
50.0
60.0
Figure 4. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of wheat straw pre-treated solids as a function of IL concentration used in the pre-treatment. The enzymatic hydrolysis yield for native wheat straw is presented for comparison. Figure 4. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of wheat straw pre-treated solids as a function of IL concentration used in the pre-treatment. The enzymatic hydrolysis yield for native wheat straw is presented for comparison. Figure 4. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of wheat straw pre-treated solids as a function of IL concentration used in the pre-treatment. The enzymatic hydrolysis yield for native wheat straw is presented for comparison. As can be observed in Figure 4, the pre-treatment of wheat straw with [emim][HSO4] allowed
the enzymatic hydrolysis of cellulose to glucose to increase by 3-fold in comparison to native biomass. As can be observed in Figure 4, the pre-treatment of wheat straw with [emim][HSO4] allowed
the enzymatic hydrolysis of cellulose to glucose to increase by 3-fold in comparison to native biomass. Interestingly, the enzymatic hydrolysis yield of cellulose (light grey bars) changed only slightly with Molecules 2019, 24, 808
Interestingly, the e 6 of 17
tly with the IL concentration used in pre-treatment and varied from 53.0 ± 0.7 mol.% to 61.6 ± 0.2 mol% for
60 and 30 wt.% of IL, respectively. On the other hand, the xylan hydrolysis yield decrease was more
pronounced, with an increase of IL concentration from 71.7 ± 0.4 mol% to 47.3 ± 1.5 mol% for 20
and 60 wt.% of IL concentration, respectively. 2.1.2. Enzymatic Hydrolysis of Pre-Treated Solids
The efficiency of pre-treatment of wheat stra
l
d b
i
h d
l
i
Th The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of eucalyptus residues pre-treated solids as a function of IL concentration used in
the pre-treatment. The enzymatic hydrolysis yield for native eucalyptus residues is presented
for comparison. u t e
o e, o euca yptus esidues p e t eated so ids, a
i c ease o I
co ce t atio
ad a
generally positive effect on the yield of enzymatic hydrolysis. For example, an enhancement of glucan
to glucose yield was found with an increase of IL concentration; however, the hydrolysis yields
detected were very low and did not exceed 14 mol%, i.e., 4.5-fold lower than observed for wheat
straw. In previous work, Xu et al. verified that the cellulose-rich solids from eucalyptus residues after
[bmim][HSO4]-catalysed hydrothermal microwave pre-treatment, allowed the achievement of
greater glucose conversion yield (89.2%). However, this was only possible when temperatures as high
as 200 °C were used in pre-treatment and enzymatic hydrolysis was performed at 2 w/v% of substrate
concentrations and 20 FPU/g substrate after 72 h [28]. Brand et al. demonstrated that the pre-
treatment time has a significant effect on the enzymatic saccharification [18]. The pre-treatment of
Miscanthus with [bmim][HSO4] at 120 °C for 8 h resulted in a solid which produced ~80% glucose and
~30% hemicellulose release. It is noteworthy that enzymatic saccharification was performed
according to very favourable NREL conditions for enzymatic hydrolysis [30]. These very different results for wheat straw and eucalyptus residues drove us to employ the
Furthermore, for eucalyptus residues pre-treated solids, an increase of IL concentration had
a generally positive effect on the yield of enzymatic hydrolysis. For example, an enhancement of
glucan to glucose yield was found with an increase of IL concentration; however, the hydrolysis yields
detected were very low and did not exceed 14 mol%, i.e., 4.5-fold lower than observed for wheat
straw. In previous work, Xu et al. verified that the cellulose-rich solids from eucalyptus residues
after [bmim][HSO4]-catalysed hydrothermal microwave pre-treatment, allowed the achievement of
greater glucose conversion yield (89.2%). However, this was only possible when temperatures as
high as 200 ◦C were used in pre-treatment and enzymatic hydrolysis was performed at 2 w/v% of
substrate concentrations and 20 FPU/g substrate after 72 h [28]. Brand et al. demonstrated that the
pre-treatment time has a significant effect on the enzymatic saccharification [18]. 2.1.2. Enzymatic Hydrolysis of Pre-Treated Solids
The efficiency of pre-treatment of wheat stra
l
d b
i
h d
l
i
Th The reason for this might be that with an increase
in the reaction intensity, more hemicellulose was extracted from the biomass, and consequently
less hemicellulose accessible for enzymatic attack was present in the pre-treated solid. In addition,
the formation of pseudo-humins may interfere with the accessibility of enzymes to hemicellulose,
and lignin presence affects an enzymatic hydrolysis because of unproductive and irreversible cellulase
enzyme adsorption on lignin [29]. and 30 wt.% of IL, respectively. On the other hand, the xylan hydrolysis yield decrease was more
pronounced, with an increase of IL concentration from 71.7 ± 0.4 mol% to 47.3 ± 1.5 mol% for 20 and
60 wt.% of IL concentration, respectively. The reason for this might be that with an increase in the
reaction intensity, more hemicellulose was extracted from the biomass, and consequently less
hemicellulose accessible for enzymatic attack was present in the pre-treated solid. In addition, the
formation of pseudo-humins may interfere with the accessibility of enzymes to hemicellulose, and
lignin presence affects an enzymatic hydrolysis because of unproductive and irreversible cellulase
enzyme adsorption on lignin [29]. Analogous to wheat straw, pre-treatment of eucalyptus residues with [emim][HSO4] improved
the enzymatic hydrolysability in comparison to native biomass as can be seen in Figure 5 Analogous to wheat straw, pre-treatment of eucalyptus residues with [emim][HSO4] improved
the enzymatic hydrolysability in comparison to native biomass as can be seen in Figure 5. the enzymatic hydrolysability in comparison to native biomass as can be seen in Figure 5. Figure 5. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of eucalyptus residues pre-treated solids as a function of IL concentration used in the pre-
treatment. The enzymatic hydrolysis yield for native eucalyptus residues is presented for comparison. IL (wt.%)
Hydrolysis yield (mol%)
0
3
6
9
12
15
native
20.0
30.0
41.3
50.0
Figure 5. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of eucalyptus residues pre-treated solids as a function of IL concentration used in
the pre-treatment. The enzymatic hydrolysis yield for native eucalyptus residues is presented
for comparison. IL (wt.%) Figure 5. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of eucalyptus residues pre-treated solids as a function of IL concentration used in the pre
treatment. The enzymatic hydrolysis yield for native eucalyptus residues is presented for comparison
Figure 5. 2.2.1. Biomass Pre-Treatment with [emim][OAc]
2.2.1. Biomass Pre-Treatment with [emim][OAc] Black bars correspond to other non-identified components and were calculated as
the difference between biomass used for process or characterised fraction and the cellulose,
hemicellulose and lignin determined. Figure 6. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from wheat straw pre-treated with [emim][OAc] at 120 ◦C and 140 ◦C and
2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours
were used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S1 in the Electronic Supplementary
Information (ESI). Black bars correspond to other non-identified components and were calculated as the
difference between biomass used for process or characterised fraction and the cellulose, hemicellulose
and lignin determined. The results obtained demonstrate that temperature has an effect on the selectivity of the
fractionation because, although a significantly lower amount of cellulose-rich fraction was obtained
at 140 °C in comparison to 120 °C (48.0 wt.% vs. 65.8 wt.% for 140 and 120 °C, respectively), it
contained more cellulose, i.e., 57.6 ± 0.3 wt.% vs. 71.4 ± 0.6 wt.% for 120 and 140 °C, respectively. However, as can be seen in Figure 6, a major reason for this was insufficient fractionation of
hemicellulose and lignin because the cellulose-rich sample obtained at 120 °C still contained 17.1 ±
3.4 and 17.2 ± 1.0 wt.% of hemicellulose and lignin, respectively. The same sample obtained at 140 °C
had much lower hemicellulose and lignin content with only 10.3 ± 1.8 wt.% of each. These relevant
differences in more selective fractionation of biomass achieved at higher temperatures also found
confirmation in the hemicellulose-rich sample. Contrary to the cellulose-rich sample, the
hemicellulose-rich fraction obtained at 140 °C contained more solid, i.e., 25.8 wt.% in comparison to
20.0 wt.% for 120 °C, and this solid was enriched in hemicellulose as it encompassed 67.8 ± 1.6 wt.%
of hemicellulose, while at 120 °C it was only 61.9 ± 1.5 wt.%. Consequently, it was predominantly
counterbalanced by a difference in lignin and other component contents found in both hemicellulose-
rich solids. 2.2.1. Biomass Pre-Treatment with [emim][OAc]
2.2.1. Biomass Pre-Treatment with [emim][OAc] 2.2.1. Biomass Pre-Treatment with [emim][OAc]
2.2.1. Biomass Pre-Treatment with [emim][OAc] The biomass pre-treatment with hydrogen-bond basic [emim][OAc] IL allowed biomass
dissolution and fractionation into cellulose-, hemicellulose- and lignin-rich fractions. For this purpose,
pre-treatment of wheat straw or eucalyptus residues in [emim][OAc] at 120 and 140 ◦C, for 2 h at
a biomass/IL ratio of 1/20 (w/w) was examined on the basis of the methodology presented in the
literature [11]. The composition of the obtained solids is given in Figures 6 and 7. The biomass pre-treatment with hydrogen-bond basic [emim][OAc] IL allowed biomass
dissolution and fractionation into cellulose-, hemicellulose- and lignin-rich fractions. For this
purpose, pre-treatment of wheat straw or eucalyptus residues in [emim][OAc] at 120 and 140 °C, for
2 h at a biomass/IL ratio of 1/20 (w/w) was examined on the basis of the methodology presented in
the literature [11]. The composition of the obtained solids is given in Figures 6 and 7. Solid composition (wt.%)
0
20
40
60
80
100
T (oC)
120
140
0
20
40
60
80
100 0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
65.8
20.0
2.4 11.8
48.0
25.8
8.3
17.5
57.6
17.1
17.2 8.1
10.8
61.9
12.7 14.7
71.4
10.3
10.3
7.9
13.1
67.8
10.2
9.0 Figure 6. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from wheat straw pre-treated with [emim][OAc] at 120 °C and 140 °C and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours were
used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S1 in the Electronic Supplementary
Information (ESI). Black bars correspond to other non-identified components and were calculated as
the difference between biomass used for process or characterised fraction and the cellulose,
hemicellulose and lignin determined. Solid composition (wt.%)
0
20
40
60
80
100
T (oC)
120
140
0
20
40
60
80
100 0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
65.8
20.0
2.4 11.8
48.0
25.8
8.3
17.5
57.6
17.1
17.2 8.1
10.8
61.9
12.7 14.7
71.4
10.3
10.3
7.9
13.1
67.8
10.2
9.0
Figure 6. 2.1.2. Enzymatic Hydrolysis of Pre-Treated Solids
The efficiency of pre-treatment of wheat stra
l
d b
i
h d
l
i
Th The pre-treatment of
Miscanthus with [bmim][HSO4] at 120 ◦C for 8 h resulted in a solid which produced ~80% glucose and
~30% hemicellulose release. It is noteworthy that enzymatic saccharification was performed according
to very favourable NREL conditions for enzymatic hydrolysis [30]. These very different results for wheat straw and eucalyptus residues drove us to employ the
approach with hydrogen-bond basic IL, namely [emim][OAc]. y
y
y
y
These very different results for wheat straw and eucalyptus residues drove us to employ the
approach with hydrogen-bond basic IL, namely [emim][OAc]. 7 of 17 Molecules 2019, 24, 808 2.2. Hydrogen-Bond Basic IL
Molecules 2019, 24, x 2.2.1. Biomass Pre-Treatment with [emim][OAc]
2.2.1. Biomass Pre-Treatment with [emim][OAc] The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from wheat straw pre-treated with [emim][OAc] at 120 ◦C and 140 ◦C and
2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours
were used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S1 in the Electronic Supplementary
Information (ESI). Black bars correspond to other non-identified components and were calculated as the
difference between biomass used for process or characterised fraction and the cellulose, hemicellulose
and lignin determined. Solid composition (wt.%) Figure 6. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from wheat straw pre-treated with [emim][OAc] at 120 °C and 140 °C and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours were
used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S1 in the Electronic Supplementary
Information (ESI). Black bars correspond to other non-identified components and were calculated as
the difference between biomass used for process or characterised fraction and the cellulose,
hemicellulose and lignin determined. Figure 6. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from wheat straw pre-treated with [emim][OAc] at 120 ◦C and 140 ◦C and
2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours
were used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S1 in the Electronic Supplementary
Information (ESI). Black bars correspond to other non-identified components and were calculated as the
difference between biomass used for process or characterised fraction and the cellulose, hemicellulose
and lignin determined. Figure 6. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from wheat straw pre-treated with [emim][OAc] at 120 °C and 140 °C and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours were
used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S1 in the Electronic Supplementary
Information (ESI). 2.2.1. Biomass Pre-Treatment with [emim][OAc]
2.2.1. Biomass Pre-Treatment with [emim][OAc] The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from eucalyptus residues pre-treated with [emim][OAc] at 120 ◦C and 140 ◦C
and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours
were used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S2 in the ESI. Black bars correspond
to other non-identified components and were calculated as the difference between biomass used for
process or characterised fraction and cellulose, hemicellulose and lignin. Figure 7. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from eucalyptus residues pre-treated with [emim][OAc] at 120 °C and 140 °C
and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same
colours were used to differentiate each individual component). Numbers presented in the figure
indicate the composition (expressed in wt.%). For exact values, refer to Table S2 in the ESI. Black bars
correspond to other non-identified components and were calculated as the difference between
biomass used for process or characterised fraction and cellulose, hemicellulose and lignin. Figure 7. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from eucalyptus residues pre-treated with [emim][OAc] at 120 ◦C and 140 ◦C
and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours
were used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S2 in the ESI. Black bars correspond
to other non-identified components and were calculated as the difference between biomass used for
process or characterised fraction and cellulose, hemicellulose and lignin. As demonstrated in Figure 7, eucalyptus residues processing with [emim][OAc] at different
temperature has almost no effect on the selectivity of biomass fractionation. An increase of
temperature by 20 °C from 120 to 140 °C, similar to wheat straw, reduced the amount of cellulose-
rich sample recovered from 66.7 wt.% to 63.5 wt.% and enhanced its purity by less than 1 wt.%. Although the trends are the same as those observed for wheat straw, the changes are negligible when
compared to those presented in Figure 6. Hemicellulose-rich fractions were recovered in very small
quantities, which made their characterisation impossible. 2.2.1. Biomass Pre-Treatment with [emim][OAc]
2.2.1. Biomass Pre-Treatment with [emim][OAc] Consequently, it can be concluded that
although biomass fractionation to cellulose-, hemicellulose- and lignin-rich fraction occurred the
eucalyptus residues make the process with hydrogen-bond basic IL less efficient than is the case for
herbaceous biomass and others presented in the literature [9,11,32,33]. As demonstrated in Figure 7, eucalyptus residues processing with [emim][OAc] at different
temperature has almost no effect on the selectivity of biomass fractionation. An increase of temperature
by 20 ◦C from 120 to 140 ◦C, similar to wheat straw, reduced the amount of cellulose-rich sample
recovered from 66.7 wt.% to 63.5 wt.% and enhanced its purity by less than 1 wt.%. Although the trends
are the same as those observed for wheat straw, the changes are negligible when compared to those
presented in Figure 6. Hemicellulose-rich fractions were recovered in very small quantities, which
made their characterisation impossible. Consequently, it can be concluded that although biomass
fractionation to cellulose-, hemicellulose- and lignin-rich fraction occurred the eucalyptus residues
make the process with hydrogen-bond basic IL less efficient than is the case for herbaceous biomass
and others presented in the literature [9,11,32,33]. 2.2.1. Biomass Pre-Treatment with [emim][OAc]
2.2.1. Biomass Pre-Treatment with [emim][OAc] Interestingly, the lignin-rich fraction obtained at 120 °C was negligible (2.4 wt.%), while
The results obtained demonstrate that temperature has an effect on the selectivity of the
fractionation because, although a significantly lower amount of cellulose-rich fraction was obtained at
140 ◦C in comparison to 120 ◦C (48.0 wt.% vs. 65.8 wt.% for 140 and 120 ◦C, respectively), it contained
more cellulose, i.e., 57.6 ± 0.3 wt.% vs. 71.4 ± 0.6 wt.% for 120 and 140 ◦C, respectively. However, as
can be seen in Figure 6, a major reason for this was insufficient fractionation of hemicellulose and lignin
because the cellulose-rich sample obtained at 120 ◦C still contained 17.1 ± 3.4 and 17.2 ± 1.0 wt.%
of hemicellulose and lignin, respectively. The same sample obtained at 140 ◦C had much lower
hemicellulose and lignin content with only 10.3 ± 1.8 wt.% of each. These relevant differences in
more selective fractionation of biomass achieved at higher temperatures also found confirmation in
the hemicellulose-rich sample. Contrary to the cellulose-rich sample, the hemicellulose-rich fraction
obtained at 140 ◦C contained more solid, i.e., 25.8 wt.% in comparison to 20.0 wt.% for 120 ◦C, and
this solid was enriched in hemicellulose as it encompassed 67.8 ± 1.6 wt.% of hemicellulose, while
at 120 ◦C it was only 61.9 ± 1.5 wt.%. Consequently, it was predominantly counterbalanced by a
difference in lignin and other component contents found in both hemicellulose-rich solids. Interestingly,
the lignin-rich fraction obtained at 120 ◦C was negligible (2.4 wt.%), while that produced at 140 ◦C Molecules 2019, 24, 808 8 of 17
8 of 17 was more than three times higher and was equal to 8.3 wt.%. This again clearly indicates that a higher
temperature was more effective in the fractionation of wheat straw and allowed the production of
cellulose-, hemicellulose- and lignin-rich fractions characterised by higher purity. allowed the production of cellulose-, hemicellulose- and lignin-rich fractions characterised by higher
purity. Labbé et al. also concluded that at high temperatures, [emim][OAc] is able to cleave the acetyl Labbé et al. also concluded that at high temperatures, [emim][OAc] is able to cleave the acetyl
groups covalently attached, mostly to the hemicellulose component of yellow poplar [31]. Therefore,
at a higher temperature this IL can effectively disrupt the carbohydrate–lignin linkages favouring
hemicellulose release. g
p
, [
][
]
y
groups covalently attached, mostly to the hemicellulose component of yellow poplar [31]. 2.2.1. Biomass Pre-Treatment with [emim][OAc]
2.2.1. Biomass Pre-Treatment with [emim][OAc] Therefore,
at a higher temperature this IL can effectively disrupt the carbohydrate–lignin linkages favouring
hemicellulose release. Figure 7. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from eucalyptus residues pre-treated with [emim][OAc] at 120 °C and 140 °C
and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same
colours were used to differentiate each individual component). Numbers presented in the figure
indicate the composition (expressed in wt.%). For exact values, refer to Table S2 in the ESI. Black bars
correspond to other non-identified components and were calculated as the difference between
biomass used for process or characterised fraction and cellulose, hemicellulose and lignin. Solid composition (wt.%)
0
20
40
60
80
100
T (oC)
120
140
0
20
40
60
80
100
0
20
40
60
80
100
66.7
4.63.8
24.8
63.5
2.2 10.5
23.8
62.4
13.9
23.1
62.7
14.4 21.9 1.0
0.7
Figure 7. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from eucalyptus residues pre-treated with [emim][OAc] at 120 ◦C and 140 ◦C
and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same colours
were used to differentiate each individual component). Numbers presented in the figure indicate the
composition (expressed in wt.%). For exact values, refer to Table S2 in the ESI. Black bars correspond
to other non-identified components and were calculated as the difference between biomass used for
process or characterised fraction and cellulose, hemicellulose and lignin. Solid composition (wt.%)
0
20
40
60
80
100
T (oC)
120
140
0
20
40
60
80
100
0
20
40
60
80
100
66.7
4.63.8
24.8
63.5
2.2 10.5
23.8
62.4
13.9
23.1
62.7
14.4 21.9 1.0
0.7 Solid composition (wt.%) Figure 7. The cellulose- (white bar), hemicellulose- (light grey bar), and Klason lignin-rich (dark grey
bar) fractions obtained from eucalyptus residues pre-treated with [emim][OAc] at 120 °C and 140 °C
and 2 h. Inserts present the composition of cellulose- and hemicellulose-rich fractions (the same
colours were used to differentiate each individual component). Numbers presented in the figure
indicate the composition (expressed in wt.%). For exact values, refer to Table S2 in the ESI. Black bars
correspond to other non-identified components and were calculated as the difference between
biomass used for process or characterised fraction and cellulose, hemicellulose and lignin. Figure 7. 2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids
2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of pre-treated wheat straw and eucalyptus residues solids produced in processes at 140 ◦C
with aqueous [emim][HSO4] (30 wt.%, 90 min) and [emim][OAc] (2 h). The enzymatic hydrolysis yield
for native biomasses is presented for comparison. The results obtained demonstrate clearly that pre-treatment of either wheat straw or eucalyptus
residues with [emim][OAc] dramatically enhanced the enzymatic hydrolysis yields. For example, for
the cellulose-rich sample of wheat straw obtained from pre-treatment with [emim][OAc], the glucan
to glucose yield was as high as 93.1 ± 4.1 mol%, while for the same biomass pre-treated with
[emim][HSO4] a maximum hydrolysis yield of 61.6 ± 0.2 mol% was achieved. Although this 50%
enhancement is remarkable, the same process for eucalyptus residues demonstrated an even more
astonishing improvement of glucan to glucose hydrolysis yield. As was shown above, the pre-treated
solids obtained after reaction with [emim][HSO4] allowed achievement of a very modest enzymatic
hydrolysis yield of 7.9 ± 0.3 mol%, but the enzymatic hydrolysis yield of cellulose-rich solid obtained
from the pre-treatment of eucalyptus residues with [emim][OAc] was as high as 82.9 ± 1.2 mol%. In
terms of potential valorisation of cellulose present in the native biomass, a switch from [emim][HSO4]
to [emim][OAc] was also demonstrated to be a better choice for both biomasses. The yield of glucose
released after both steps (pre-treatment with [emim][OAc] and posterior enzymatic hydrolysis) was
as high as 91.4 mol% and 74.9 mol% for wheat straw and eucalyptus residues, respectively. The same
yield for both biomasses pre-treated with [emim][HSO4] with 30 wt.% of IL was only 62.3 mol% and
7.9 mol% for wheat straw and eucalyptus, respectively. The results obtained demonstrate clearly that pre-treatment of either wheat straw or eucalyptus
residues with [emim][OAc] dramatically enhanced the enzymatic hydrolysis yields. For example, for
the cellulose-rich sample of wheat straw obtained from pre-treatment with [emim][OAc], the glucan
to glucose yield was as high as 93.1 ± 4.1 mol%, while for the same biomass pre-treated with
[emim][HSO4] a maximum hydrolysis yield of 61.6 ± 0.2 mol% was achieved. Although this 50%
enhancement is remarkable, the same process for eucalyptus residues demonstrated an even more
astonishing improvement of glucan to glucose hydrolysis yield. 2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids
2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids As was shown above, the pre-treated
solids obtained after reaction with [emim][HSO4] allowed achievement of a very modest enzymatic
hydrolysis yield of 7.9 ± 0.3 mol%, but the enzymatic hydrolysis yield of cellulose-rich solid obtained
from the pre-treatment of eucalyptus residues with [emim][OAc] was as high as 82.9 ± 1.2 mol%. In terms of potential valorisation of cellulose present in the native biomass, a switch from [emim][HSO4]
to [emim][OAc] was also demonstrated to be a better choice for both biomasses. The yield of glucose
released after both steps (pre-treatment with [emim][OAc] and posterior enzymatic hydrolysis) was as
high as 91.4 mol% and 74.9 mol% for wheat straw and eucalyptus residues, respectively. The same
yield for both biomasses pre-treated with [emim][HSO4] with 30 wt.% of IL was only 62.3 mol% and
7.9 mol% for wheat straw and eucalyptus, respectively. 7.9 mol% for wheat straw and eucalyptus, respectively. These results indicate that a change of the IL used, from hydrogen-bond acidic to hydrogen-
bond basic IL, promoted a significant change in the pre-treated solids, as the enzymatic hydrolysis
yield increased by more than 1000%. The results in the literature also confirm similar, although not
such pronounced, differences. For example, Bian et al. studied the effect of IL pre-treatment on
enzymatic hydrolysis of cellulose as a function of chemical and physical structure changes [34]. In a
case of cellulose isolated from sugarcane bagasse subjected to IL ([emim][OAc]), dissolution at a mild
temperature (90 °C) followed by a solid regeneration in water, resulted in an increase in glucose
content from 80.0–83.3 wt.% to 91.6–92.8 wt.%, a decrease in the degree of polymerisation from 974–
1039 units to 511–521 units, a transformation from cellulose I to cellulose II, and an increase of surface
area during the pre-treatment. Such cellulose was subsequently hydrolysed by commercial cellulases
with 2 w/v% cellulose substrate and enzyme loadings of 35 FPU/g (cellulase) and 40 CbU (β-
These results indicate that a change of the IL used, from hydrogen-bond acidic to hydrogen-bond
basic IL, promoted a significant change in the pre-treated solids, as the enzymatic hydrolysis yield
increased by more than 1000%. The results in the literature also confirm similar, although not such
pronounced, differences. For example, Bian et al. studied the effect of IL pre-treatment on enzymatic
hydrolysis of cellulose as a function of chemical and physical structure changes [34]. 2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids
2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids y
y
y
Regardless the efficiency of both biomass fractionation processes, cellulose-rich samples were
subject to the enzymatic hydrolysis according to the method presented in the experimental section. The obtained results are presented in Figure 8 and are compared to those achieved for hydrogen-
bond acidic IL
Regardless the efficiency of both biomass fractionation processes, cellulose-rich samples were
subject to the enzymatic hydrolysis according to the method presented in the experimental section. The obtained results are presented in Figure 8 and are compared to those achieved for hydrogen-bond
acidic IL. 9 of 17
9 of 17 Molecules 2019, 24, 808
Molecules 2019, 24, x Figure 8. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of pre-treated wheat straw and eucalyptus residues solids produced in processes at 140 °C
with aqueous [emim][HSO4] (30 wt.%, 90 min) and [emim][OAc] (2 h). The enzymatic hydrolysis yield
for native biomasses is presented for comparison. Hydrolysis yield (mol%)
0
10
20
60
80
100
native
[emim][HSO4]
[emim][OAc]
native
wheat straw
eucalyptus
[emim][HSO4]
[emim][OAc]
Figure 8. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of pre-treated wheat straw and eucalyptus residues solids produced in processes at 140 ◦C
with aqueous [emim][HSO4] (30 wt.%, 90 min) and [emim][OAc] (2 h). The enzymatic hydrolysis yield
for native biomasses is presented for comparison. eucalyptus wheat straw Figure 8. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of pre-treated wheat straw and eucalyptus residues solids produced in processes at 140 °C
with aqueous [emim][HSO4] (30 wt.%, 90 min) and [emim][OAc] (2 h). The enzymatic hydrolysis yield
for native biomasses is presented for comparison. Figure 8. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of pre-treated wheat straw and eucalyptus residues solids produced in processes at 140 ◦C
with aqueous [emim][HSO4] (30 wt.%, 90 min) and [emim][OAc] (2 h). The enzymatic hydrolysis yield
for native biomasses is presented for comparison. Figure 8. The enzymatic hydrolysis yield (glucan to glucose–light grey bar; and xylan to xylose–dark
grey bar) of pre-treated wheat straw and eucalyptus residues solids produced in processes at 140 °C
with aqueous [emim][HSO4] (30 wt.%, 90 min) and [emim][OAc] (2 h). The enzymatic hydrolysis yield
for native biomasses is presented for comparison. Figure 8. 2.3. Morphological Analysis of Pre-Treated Solids
2.3. Morphological Analysis of Pre Treated Solid
Crystalline cellulose is the most orga Crystalline cellulose is the most organised form of cellulose in the biomass [36]. Also, crystallinity
of cellulose has been reported as one of the most relevant factors influencing the efficiency of enzymatic
hydrolysis [37]. X-ray diffraction permits measurement of the crystallinity of the material as a whole,
because it demonstrates either crystalline (organised) or disordered components of the biomass, namely,
amorphous cellulose, hemicellulose and lignin [38]. ILs have been shown to affect cellulose crystallinity
during pre-treatment and consequently enhancing the enzymatic hydrolysis [39]. Therefore, the effect
of different pre-treatment approaches on crystallinity, which could justify the efficiency of enzymatic
hydrolysis, was also tested in this work. The results obtained for wheat straw and eucalyptus residues
are depicted in Figures 9 and 10, respectively. y
g
[
]
,
crystallinity of cellulose has been reported as one of the most relevant factors influencing the
efficiency of enzymatic hydrolysis [37]. X-ray diffraction permits measurement of the crystallinity of
the material as a whole, because it demonstrates either crystalline (organised) or disordered
components of the biomass, namely, amorphous cellulose, hemicellulose and lignin [38]. ILs have
been shown to affect cellulose crystallinity during pre-treatment and consequently enhancing the
enzymatic hydrolysis [39]. Therefore, the effect of different pre-treatment approaches on crystallinity,
which could justify the efficiency of enzymatic hydrolysis, was also tested in this work. The results
obtained for wheat straw and eucalyptus residues are depicted in Figures 9 and 10, respectively. Figure 9. Powder XRD patterns of: (a) native wheat straw, (b) pre-treated with [emim][HSO4], and (c)
pre-treated with [emim][OAc] IL. 2Θ
10
20
30
40
Arbitrary Units
a)
b)
c)
Figure 9. Powder XRD patterns of: (a) native wheat straw, (b) pre-treated with [emim][HSO4], and
(c) pre-treated with [emim][OAc] IL. Figure 9. Powder XRD patterns of: (a) native wheat straw, (b) pre-treated with [emim][HSO4], and (c)
pre-treated with [emim][OAc] IL. Figure 9. Powder XRD patterns of: (a) native wheat straw, (b) pre-treated with [emim][HSO4], and
(c) pre-treated with [emim][OAc] IL. Figures 9a and 10a show the diffraction patterns of untreated wheat straw and eucalyptus
residues. The main signal can be observed at 22.3° for wheat straw and at 22.5° for eucalyptus residue
samples. This signal indicates the distance between hydrogen-bonded sheets in cellulose I and
corresponds to the (200) lattice plane. 2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids
2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids 2 3 M
h l
i
l A
l
i
f P
T
t d S lid (β-glucosidase) in relation to the dry weight of cellulose substrates, and allowed achievement of a high
glucose conversion yield of 95.2 mol%. These results suggest that pre-treatment led to an effective
disruption of cellulose favouring enzyme hydrolysis. Torr et al. also observed an improvement of
the glucan to glucose yield after saccharification for 72 h performed at biomass loading of 1.5% (w/v)
and Celluclast 1.5 L and Novozymes 188 with 40 FPUs/g glucan and β-glucosidades of 50 IU/g
glucan, from 5 mol% in the untreated pine wood to 84 mol% in wood pre-treated with [emim][OAc]. The analysis of the substrates revealed that the most important change brought by the pre-treatment
was an increase in the accessible surface area. In this case, the delignification was not observed, and loss
of cellulose crystallinity only occurred for the highest intensity pre-treatments [35]. Therefore, to verify
this, changes in the morphology of pre-treated solid materials were studied using X-ray diffraction. g
y
gg
p
disruption of cellulose favouring enzyme hydrolysis. Torr et al. also observed an improvement of the
glucan to glucose yield after saccharification for 72 h performed at biomass loading of 1.5% (w/v) and
Celluclast 1.5 L and Novozymes 188 with 40 FPUs/g glucan and β-glucosidades of 50 IU/g glucan,
from 5 mol% in the untreated pine wood to 84 mol% in wood pre-treated with [emim][OAc]. The
analysis of the substrates revealed that the most important change brought by the pre-treatment was
an increase in the accessible surface area. In this case, the delignification was not observed, and loss
of cellulose crystallinity only occurred for the highest intensity pre-treatments [35]. Therefore, to
verify this, changes in the morphology of pre-treated solid materials were studied using X-ray
diffraction. 2 3 Morphological Analysis of Pre Treated Solids 2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids
2.2.2. Enzymatic Hydrolysis of Pre-Treated Solids In a case
of cellulose isolated from sugarcane bagasse subjected to IL ([emim][OAc]), dissolution at a mild
temperature (90 ◦C) followed by a solid regeneration in water, resulted in an increase in glucose
content from 80.0–83.3 wt.% to 91.6–92.8 wt.%, a decrease in the degree of polymerisation from
974–1039 units to 511–521 units, a transformation from cellulose I to cellulose II, and an increase of
surface area during the pre-treatment. Such cellulose was subsequently hydrolysed by commercial
cellulases with 2 w/v% cellulose substrate and enzyme loadings of 35 FPU/g (cellulase) and 40 CbU 10 of 17
10 of 17 Molecules 2019, 24, 808
Molecules 2019, 24, x (β-glucosidase) in relation to the dry weight of cellulose substrates, and allowed achievement of a high
glucose conversion yield of 95.2 mol%. These results suggest that pre-treatment led to an effective
disruption of cellulose favouring enzyme hydrolysis. Torr et al. also observed an improvement of
the glucan to glucose yield after saccharification for 72 h performed at biomass loading of 1.5% (w/v)
and Celluclast 1.5 L and Novozymes 188 with 40 FPUs/g glucan and β-glucosidades of 50 IU/g
glucan, from 5 mol% in the untreated pine wood to 84 mol% in wood pre-treated with [emim][OAc]. The analysis of the substrates revealed that the most important change brought by the pre-treatment
was an increase in the accessible surface area. In this case, the delignification was not observed, and loss
of cellulose crystallinity only occurred for the highest intensity pre-treatments [35]. Therefore, to verify
this, changes in the morphology of pre-treated solid materials were studied using X-ray diffraction. g
y
gg
p
disruption of cellulose favouring enzyme hydrolysis. Torr et al. also observed an improvement of the
glucan to glucose yield after saccharification for 72 h performed at biomass loading of 1.5% (w/v) and
Celluclast 1.5 L and Novozymes 188 with 40 FPUs/g glucan and β-glucosidades of 50 IU/g glucan,
from 5 mol% in the untreated pine wood to 84 mol% in wood pre-treated with [emim][OAc]. The
analysis of the substrates revealed that the most important change brought by the pre-treatment was
an increase in the accessible surface area. In this case, the delignification was not observed, and loss
of cellulose crystallinity only occurred for the highest intensity pre-treatments [35]. Therefore, to
verify this, changes in the morphology of pre-treated solid materials were studied using X-ray
diffraction. 2.3. Morphological Analysis of Pre-Treated Solids
2.3. Morphological Analysis of Pre Treated Solid
Crystalline cellulose is the most orga For both biomasses, the second main band observed is a
broad signal registered at 2θ = 16.7° and corresponds to overlapping signals of (101) and (10-1
Figures 9a and 10a show the diffraction patterns of untreated wheat straw and eucalyptus residues. The main signal can be observed at 22.3◦for wheat straw and at 22.5◦for eucalyptus residue samples. This signal indicates the distance between hydrogen-bonded sheets in cellulose I and corresponds to
the (200) lattice plane. For both biomasses, the second main band observed is a broad signal registered
at 2θ = 16.7◦and corresponds to overlapping signals of (101) and (10-1) planes. The “valley” at 18.1◦is 11 of 17
11 of 17 Molecules 2019, 24, 808
Molecules 2019, 24, x associated with an amorphous region in the biomass and includes disordered cellulose, hemicellulose
and lignin. The third, barely noticeable, signal at 34.5◦corresponds to one-quarter of the length of one
cellobiose unit and arises from ordering along the fibre direction. disordered cellulose, hemicellulose and lignin. The third, barely noticeable, signal at 34.5°
corresponds to one-quarter of the length of one cellobiose unit and arises from ordering along the
fibre direction. Figure 10. Powder XRD patterns of: (a) native eucalyptus residues, (b) pre-treated with [emim][HSO4]
and (c) pre-treated with [emim][OAc] IL. 2Θ
10
20
30
40
Arbitrary Units
a)
b)
c)
Figure 10. Powder XRD patterns of: (a) native eucalyptus residues, (b) pre-treated with [emim][HSO4]
and (c) pre-treated with [emim][OAc] IL. 2Θ Figure 10. Powder XRD patterns of: (a) native eucalyptus residues, (b) pre-treated with [emim][HSO4]
and (c) pre-treated with [emim][OAc] IL. Figure 10. Powder XRD patterns of: (a) native eucalyptus residues, (b) pre-treated with [emim][HSO4]
and (c) pre-treated with [emim][OAc] IL. The XRD patterns of wheat straw and eucalyptus residues pre-treated with [emim][HSO4]
indicated in Figures 9b and 10b as diffractograms, respectively, mirror the diffractograms observed
for native biomasses (Figures 9a and 10a). This confirms that hydrogen-bond acidic ILs, such as
[emim][HSO4] tested in this work, do not induce any qualitative changes in the pre-treated samples. The same cannot be said about the cellulose-rich solids produced in the [emim][OAc] pre-treatment. The most dominant change is a disappearance of signal at 22.5°, and the presence of a broad
asymmetric signal consisting of a doublet at 20° and 21.7°, as demonstrated in Figure 9c. 2.3. Morphological Analysis of Pre-Treated Solids
2.3. Morphological Analysis of Pre Treated Solid
Crystalline cellulose is the most orga Similarly,
the broad signal at 16° disappeared and was substituted with a new signal that emerged at 12.1°, as
can be seen in the same figure. These changes are characteristic of a transformation of cellulose I to
cellulose II and they are the most visible for wheat straw pre-treated samples. For the cellulose-rich
sample of eucalyptus residues, similar changes in the diffractogram are visible although they are less
notable. For example, as shown in Figure 10c, a broad signal at 20–22° can be found. Additionally, a
signal at 16° became very wide and was transformed into the arm of the main signal. These changes
in signals confirm the alteration in the cellulose organisation similar to what was observed for wheat
straw. At the same time, as these signals are still not complete defined, contrary to what was observed
for the wheat straw cellulose-rich sample, it may indicate that in case of eucalyptus residues, the
transformation of crystalline cellulose is less effective and may require longer pre-treatment time. Regardless of the reasons, the observed changes in the XRD patterns justify the fact that even partial
alteration of biomass crystallinity is sufficient to promote more efficient enzymatic hydrolysis, as
demonstrated in this work. Similar results were presented in the literature, where it was confirmed
that cellulose II is more readily digested than cellulose I. It has been argued that the van der Waals
interactions between hydrogen-bonded sheets in cellulose I are stronger than in cellulose II and that
they act as the main factor in resisting cellulose hydrolysis [40] Li et al compared the pre-treatment
The XRD patterns of wheat straw and eucalyptus residues pre-treated with [emim][HSO4]
indicated in Figures 9b and 10b as diffractograms, respectively, mirror the diffractograms observed
for native biomasses (Figures 9a and 10a). This confirms that hydrogen-bond acidic ILs, such as
[emim][HSO4] tested in this work, do not induce any qualitative changes in the pre-treated samples. The same cannot be said about the cellulose-rich solids produced in the [emim][OAc] pre-treatment. The most dominant change is a disappearance of signal at 22.5◦, and the presence of a broad asymmetric
signal consisting of a doublet at 20◦and 21.7◦, as demonstrated in Figure 9c. Similarly, the broad signal
at 16◦disappeared and was substituted with a new signal that emerged at 12.1◦, as can be seen in the
same figure. 3.1. Materials The wheat straw sample was delivered by ECN (Energy Research Centre of the Netherlands,),
from Petten, the Netherlands. The eucalyptus residues were kindly provided by The Navigator
Company from their paper mill in Cacia, Portugal. The wheat straw and eucalyptus residues moisture
content was found to be 9.8 and 8.4% (w/w), respectively and was determined using an AMB-50
moisture analyser. Both feedstocks were ground with a knife mill IKA® WERKE, MF 10 basic (Staufen, Germany) to
particles smaller than 0.5 mm, homogenised in a defined lot, and stored in plastic containers at room
temperature prior to further use. The [emim][HSO4] IL (99 wt.% of purity) was purchased from Iolitec GmBH—Heilbronn,
Germany and was used in reactions without any previous purification. The water content in IL
was determined by a volumetric Karl-Fischer titration and was 3796 ppm. The [emim][OAc] with
stated purity >95% was purchased from Iolitec GmbH—Heilbronn, Germany. Prior to use in the
pre-treatment, this IL was subject to drying under vacuum (0.1 Pa) at room temperature for at least
24 h. The water content in this IL was 2800 ppm, determined by a volumetric Karl-Fischer titration as
for the other IL. In pre-treatment experiments, the following reagents were used:
0.1 M and 3% (w/w)
NaOH aqueous solutions prepared from NaOH pellets (99% purity) supplied by Eka chemicals/
Akzonobel–Bohus, Sweden. The aqueous solutions of 1 M and 4 M HCl were prepared from fuming
HCl 37% (w/w) with a purity grade for analysis (Merck—Darmstadt, Germany). Ethanol 96% (v/v) and
acetonitrile of HPLC-gradient purity for analysis (Carlo Erba Group—Arese, Italy) and acetone (98%
purity) were supplied by Valente & Ribeiro, Lda.—Belas, Portugal. For the preparation of NaOH and
HCl solutions, distilled water (17 M Ωcm−1) and ultrapure water (18.2 MΩcm−1) both produced by
the PURELAB Classic of Elga system were used. For filtration, paper and glass microfibre filters (Whatman GE Healthcare Bio-Sciences
Corp.—Piscataway, NJ, USA) and nylon filters, 0.45 lm HNPW (Merck Millipore—Billerica, MA,
USA) were used. Glucose (≥98 wt.%, Merck—Darmstadt, Germany), xylose (≥98 wt.%, Merck, Germany), arabinose
(≥98 wt.%, Merck, Germany), furfural (furan-2-carbaldehyde) (99 wt.%, Sigma-Aldrich—Steinheim,
Germany), 5-hydroxymethylfurfural (5-(hydroxymethyl)-2-furaldehyde) (99 wt.%, Sigma-Aldrich,
Germany) and acetic acid (glacial, 99.8 wt.% Merck—Darmstadt, Germany) were used for the
qualitative and quantitative HPLC analyses of the obtained liquids and solids. Sulphuric acid (96 wt.%,
Panreac—Castellar del Vallès, Spain) was used to prepare mobile phase for HPLC analyses (5 mM
sulphuric acid). 2.3. Morphological Analysis of Pre-Treated Solids
2.3. Morphological Analysis of Pre Treated Solid
Crystalline cellulose is the most orga These changes are characteristic of a transformation of cellulose I to cellulose II and they
are the most visible for wheat straw pre-treated samples. For the cellulose-rich sample of eucalyptus
residues, similar changes in the diffractogram are visible although they are less notable. For example,
as shown in Figure 10c, a broad signal at 20–22◦can be found. Additionally, a signal at 16◦became
very wide and was transformed into the arm of the main signal. These changes in signals confirm the
alteration in the cellulose organisation similar to what was observed for wheat straw. At the same
time, as these signals are still not complete defined, contrary to what was observed for the wheat
straw cellulose-rich sample, it may indicate that in case of eucalyptus residues, the transformation
of crystalline cellulose is less effective and may require longer pre-treatment time. Regardless of
the reasons, the observed changes in the XRD patterns justify the fact that even partial alteration of
biomass crystallinity is sufficient to promote more efficient enzymatic hydrolysis, as demonstrated in
this work. Similar results were presented in the literature, where it was confirmed that cellulose II is
more readily digested than cellulose I. It has been argued that the van der Waals interactions between
hydrogen-bonded sheets in cellulose I are stronger than in cellulose II and that they act as the main
factor in resisting cellulose hydrolysis [40]. Li et al. compared the pre-treatment of switchgrass with 12 of 17 Molecules 2019, 24, 808 [emim][OAc] and with a 1.2% (w/w) dilute sulphuric acid [41]. For both untreated and dilute acid
pre-treated switchgrass samples, little or no change in cellulose crystallinity was observed, but for the
sample obtained after pre-treatment with [emim][OAc] the crystallinity was altered significantly, with
a structural transformation from cellulose I to cellulose II observed. This, in turn, promoted better
performance of enzymatic hydrolysis. 3.3.1. Pre-Treatment of Biomass with [emim][HSO4] All reactions were performed with a 10 wt.% of dry biomass in the reaction mixture. For this
purpose, 0.5 g of dry biomass and 4.5 g of aqueous IL solution with different [emim][HSO4]/H2O
ratios, were placed into a 15 mL glass vial (Supelco/Sigma-Aldrich, Bellefonte, PA, USA). Next, a vial
was placed into the oil bath pre-heated to the desired temperature (140 ◦C), and reactions were carried
out for 90 min, under continuous magnetic stirring. The reaction condition was selected on the basis
of previously published work [17]. After reaction, the mixture was cooled to room temperature
and approximately 5.0 mL of ultrapure H2O was added to precipitate non-hydrolysed fractions. The resulting mixture was filtered under vacuum using 0.45 µm nylon membrane filters. The liquid
phase was collected and stored in a freezer for posterior analysis by HPLC, while recovered solid
biomass was washed with 100 mL of ultrapure H2O (in 10 mL portions) to guarantee removal of IL
from the precipitated solid. Next, the obtained solid was placed in the oven at 60 ◦C for 24 h and
afterwards was stored at room temperature for 1 h to analyse the dry mass content. The composition
of the solid was analysed as presented in Section 2.2. 3.1. Materials For the enzymatic hydrolysis assays, 0.1 M sodium citrate buffer (pH 4.8) prepared from citric acid
monohydrate (99.7% purity) and tris-sodium citrate (>99% purity) both from VWR International
Ltd.—Leicester, England and a 2 wt.% sodium azide solution were used. Celli®CTec2 enzyme
solution kindly provided by Novozymes A/S Europe—Bagsvaerd, Denmark was employed in the
enzymatic reaction. Molecules 2019, 24, 808 13 of 17 13 of 17 3.2. Biomass and Pre-Treated Solid Characterisation 3.2. Biomass and Pre-Treated Solid Characterisation 3.2. Biomass and Pre-Treated Solid Characterisation Both biomasses and pre-treated solids were characterised to determine the total moisture [42],
total lignin and polysaccharide contents [27]. Acid-insoluble lignin was determined gravimetrically,
while acid-soluble lignin was established using UV spectrophotometry. The content of glucan and
hemicelluloses (xylan, arabinan, and acetyl groups) was determined using high performance liquid
chromatography (HPLC) equipment. Furthermore, for native biomasses, total extractives, ash and
protein contents were determined according to standard methods, namely: NREL/TP-510-42619 [43],
NREL/TP-510-42622 [44] and ISO 8968-1:2014 [45], respectively. p
y
The composition of both biomasses is presented in Table 1. Table 1. Wheat straw and eucalyptus residues macromolecular composition. Table 1. Wheat straw and eucalyptus residues macromolecular composition. Components (Dry Weight %)
Wheat Straw
Eucalyptus Residues
Glucan
35.9 ± 0.3
44.1 ± 0.9
Hemicellulose
26.7
19.6
Xylan
22.1 ± 0.6
15.7 ± 0.2
Arabinosyl group
2.0 ± 0.7
0.5 ± 0.1
Acetyl group
2.6 ± 0.9
3.4 ± 0.9
Lignin
16.7
33.8
Acid-insoluble
15.5 ± 0.4
26.4 ± 0.1
Acid-soluble
1.2 ± 0.1
7.4 ± 0.1
Ash
11.4 ± 0.1
1.0 ± 0.1
Extractives
Water
9.4 ± 1.3
3.3 ± 0.4
Water (not ash)
5.1 ± 0.5
0.2 ± 0.0
Ethanol
1.4 ± 0.1
1.5 ± 0.1 3.3. Biomass Processing 3.3.1. Pre-Treatment of Biomass with [emim][HSO4] 3.3.2. Pre-Treatment of Biomass with [emim][OAc] The pre-treatment was performed according to a method presented in the literature [11]. Reactions
were performed at two different temperatures (120 or 140 ◦C) for 2 h with a 5% (w/w) biomass/IL
ratio in a 15 mL vial. Following the aforementioned procedure presented in the literature, three solid
fractions, rich in cellulose, hemicellulose and lignin were obtained. All of them were characterised
according to methods presented in Section 2.2. Molecules 2019, 24, 808 14 of 17 3.5.1. HPLC Analysis The liquid phases obtained from the pre-treatment of biomass with [emim][HSO4] and enzymatic
hydrolyses, as well as from native or pre-treated biomass characterisation were analysed using Agilent
110 series equipment with a Bio-Rad Aminex HPX-87H column (Hercules, CA, USA). Analyses were
performed at 65 ◦C with 5 mmol·L−1 H2SO4 used as the mobile phase at a flow rate of 0.6 mL·min−1. The detection was performed using RID (refractive index detector) for monosaccharides (glucose,
xylose and arabinose) and acetic acid and DAD (diode array detector) at 280 nm wavelength for
furans (furfural and 5-HMF ≡5-hydroxymethylfurfural). The quantitative analyses were performed
by external calibration using standard solutions. 3.4. Enzymatic Hydrolysis of Solids The digestibility of pre-treated solids obtained with [emim][HSO4] or [emim][OAc] was evaluated
by enzymatic hydrolysis. The assays were performed using 5% (w/v) solids (dry weight basis)
concentration in 50 mL vials with 5 mL of 0.05 M sodium citrate buffer (pH 5), prepared from citric
acid monohydrate and tris-sodium citrate and 100 µL of a 2 wt.% sodium azide solution to prevent
undesired growth of microorganisms. Distilled water was added to reach 5.0 mL taking into account
the volume of enzyme added last. The enzyme dosage used was 10% (w/w cellulose) of Celli®CTec2
(199.9 FPU·mL−1). The enzymatic hydrolyses were performed in a shaking incubator (Optic Ivymen
system—Madrid, Spain) at 180 rpm and 50 ◦C for 72 h. After hydrolysis, enzymes were inactivated
by freezing the samples. To measure monosaccharide content, the hydrolysates were filtered under
vacuum using nylon filters (pore size of 0.45 µm) and analysed by HPLC. The glucose and xylose yields
were calculated considering the glucan and xylan contents and factors of (162/180) and (132/150) for
dehydration, respectively. 3.5. Chemical Analysis 3.5.2. XRD Measurements The crystallinity analyses of untreated biomasses and pre-treated materials were performed by
X-ray powder diffraction (XRD). For this purpose, a Rigaku Geigerflex D/MAX-III C X-ray powder
diffractometer with vertical goniometer, Bragg-Brentano geometry and graphite monochromator was
used. The CuKα radiation (λ = 1.5418 Å) at 45 kV and 20 mA was used. The samples were analysed in
the range of 2θ from 5◦to 50◦with increments of 0.02◦. 3.6. Experimental Uncertainty Each weighing was made with a standard uncertainty (u) u(m) of 0.1 mg. All pretreatment
experiments were performed with a u(T) of 2 ◦C. All enzymatic hydrolysis experiments were performed
with a u(T) of 0.1 ◦C. All other experimental errors related to measurements depended on the calibration
technique used to quantify the concentrations of products. All reactions (pre-treatments and enzymatic
hydrolyses) and analyses were performed in duplicate and results are given as mean values with the
corresponding standard deviation. 4. Conclusions The present work takes a major step towards providing a comparative framework between two
IL-type pre-treatments coupled with enzymatic saccharification, in terms of their performance on
converting wheat straw and eucalyptus residues to fermentable sugars. The first strategy relied
on the processing of biomass with hydrogen-bond acidic IL. This approach allowed integration of
biomass pre-treatment, hydrolysis and conversion of biomass in a single-step process. The acidic
character of the [HSO4] anion of IL promoted a selective processing of hemicellulose fraction and the
resulting products (mainly pentoses and furfural) were kept in a liquid phase. The solids produced 15 of 17 Molecules 2019, 24, 808 were mainly constituted of cellulose and lignin. The second approach with hydrogen-bond basic IL,
allowed biomass dissolution and fractionation into cellulose-, hemicellulose- and lignin-rich fractions. In this case, the recalcitrance of the lignocellulosic matrix was overcome by alteration of the strong
network of intra- and intermolecular bonds existing in the biomass. Consequently, a loss of native
cellulose crystalline I structure was observed, and the cellulose II form was obtained, which was not
observed for solids produced by [emim][HSO4]. This, in turn allowed a significant improvement in
the enzymatic hydrolysis yields to be obtained. Supplementary Materials: The following are available online: Table S1. Composition of cellulose- and
hemicellulose-rich fractions obtained from wheat straw pre-treated with [emim][OAc] at 120 and 140 ◦C and 2 h;
Table S2. Composition of cellulose-rich fractions obtained from eucalyptus pre-treated with [emim][OAc] at 120
and 140 ◦C and 2 h. Author Contributions:
Conceptualisation and Methodology, J.R.B. and R.M.Ł.; Writing—Original Draft
Preparation, J.R.B.; Writing—Review & Editing, R.M.Ł.; Funding Acquisition, F.M.G. and R.M.Ł. Author Contributions:
Conceptualisation and Methodology, J.R.B. and R.M.Ł.; Writing—Original Draft
Preparation, J.R.B.; Writing—Review & Editing, R.M.Ł.; Funding Acquisition, F.M.G. and R.M.Ł. Funding: This research was funded under the AMBITION (Advanced biofuel production with energy system
integration work) project funded by the European H2020-programme under the LCE-33 2016 European Common
Research and Innovation Agendas (ECRIAs) in support of the implementation of the SET Action Plan, Grant
Agreement 731263. This work was also supported by the Fundação para a Ciência e a Tecnologia (FCT, Portugal)
through BBRI - Biomass and Bioenergy Research Infrastructure (ROTEIRO/0189/2013) and grant IF/00471/2015
(RML). The authors also wish to thank Teresa Diamantino and Isabel Figueira (LRM, UER, LNEG) for XRD
measurements. 4. Conclusions Finally, the authors also wish to thank Susana Marques (UB, LNEG) for conceptualisation of the
enzymatic hydrolysis assays as well as Maria do Céu Penedo (UB, LNEG) for assistance in the HPLC analyses. Conflicts of Interest: The authors declare no conflict of interest. References 1. Sun, S.; Sun, S.; Cao, X.; Sun, R. The role of pretreatment in improving the enzymatic hydrolysis of
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Anti-angiogenic agents in the age of resistance to immune checkpoint inhibitors: Do they have a role in non-oncogene-addicted non-small cell lung cancer?
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A R T I C L E I N F O Keywords:
Anti-angiogenic drug
Immunotherapy resistance
Nintedanib
Non-oncogene-addicted non-small cell lung
cancer (NSCLC)
Tumor microenvironment (TME)
Vascular endothelial growth factor (VEGF) The introduction of licensed front-line immunotherapies has heralded a new era for the treatment of non-on-
cogene-addicted, advanced non-small cell lung cancer (NSCLC). Yet as with all evolutions in clinical manage-
ment, changes in practice can outpace the availability of the clinical evidence needed to inform subsequent
therapeutic decision making. At the time of writing, there is limited available evidence on the optimum ther-
apeutic options after progression on immunotherapy. Further research is needed to define mechanisms of im-
munotherapy resistance in patients with advanced NSCLC, and to understand the implications for subsequent
treatment response. Pending the availability of robust clinical data and proven therapeutic options to underpin
an optimized therapeutic pathway after progression on immunotherapy, attention must turn to the potential
utility of currently licensed agents and any available supporting clinical data in this setting. Within this context
we review the mechanistic arguments and supporting evidence for the use of anti-angiogenic agents as a means
of targeting immunosuppression within the tumor microenvironment. We consider whether VEGF inhibition
may help to normalize the tumor vasculature and to address immunosuppression – reinstating, and potentially
enhancing, the effect of subsequent therapies. We also highlight evidence needs and signpost ongoing trials that
should enable current clinical opinion in this area to be replaced by robust, evidence-based guidance. Anti-angiogenic agents in the age of resistance to immune checkpoint
inhibitors: Do they have a role in non-oncogene-addicted non-small cell lung
cancer? Sanjay Popata,b,*, Christian Grohéc, Jesus Corrald, Martin Recke, Silvia Novellof, Maya Gottfriedg,
Dejan Radonjich, Rolf Kaiserh,i a Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, Chelsea, London, SW3 6JJ, UK
b h
f
h
ld
d
d a Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, Chelsea, London, SW3 6JJ, UK
b The Institute of Cancer Research, 15 Cotswold Road, Sutton, London, SM2 5NG, UK
c Department of Respiratory Diseases, ELK, 13125, Berlin, Germany
d Clínica Universidad de Navarra en Madrid, Calle Marquesado de Sta. Marta, 1, 28027 Madrid, Spain
e Department of Thoracic Oncology, Airway Research Center North (ARCN) Member of the German Center for Lung Research (DZL), LungenClinic, Wöhrendamm 8
22927 Großhansdorf, Germany a Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, Chelsea, London, SW3 6JJ, UK
b The Institute of Cancer Research, 15 Cotswold Road, Sutton, London, SM2 5NG, UK
c Department of Respiratory Diseases, ELK, 13125, Berlin, Germany
d Clínica Universidad de Navarra en Madrid, Calle Marquesado de Sta. Marta, 1, 28027 Madrid, Spain
e Department of Thoracic Oncology, Airway Research Center North (ARCN) Member of the German Center for Lung Research (DZL), LungenClinic, Wöhrendamm 80,
22927 Großhansdorf, Germany b The Institute of Cancer Research, 15 Cotswold Road, Sutton, London, SM2 5N
c Department of Respiratory Diseases, ELK, 13125, Berlin, Germany f Department of Oncology, University of Turin, San Luigi Hospital, Regione Gonzole, 10, 10043 Orbassano TO, Turin, Italy
g Meir Medical Center, Tchernichovsky St 59, Kefar Sava, 4428164, Israel h Boehringer Ingelheim International GmbH, Binger Strasse 173, 55216 Ingelheim am Rhein, Germany
i I
tit t
f Ph
l
J h
G t
b
U i
it
M i
55122 M i
G h Boehringer Ingelheim International GmbH, Binger Strasse 173, 55216 Ingelheim am Rhein, Germ
i Institute of Pharmacology, Johannes Gutenberg-University Mainz, 55122 Mainz, Germany i Institute of Pharmacology, Johannes Gutenberg-University Mainz, 55122 Mainz, Germany Received 12 January 2020; Received in revised form 1 April 2020; Accepted 9 April 2020
0169-5002/ ©
2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecom
m
ons.org/licenses/BY/4.0/). Lung Cancer 144 (2020) 76–84 Lung Cancer 144 (2020) 76–84 Contents lists available at ScienceDirect A R T I C L E I N F O Abbreviations: Ab, antibody; ABC, atezolizumab, bevacizumab and chemotherapy combination; ANG2, angiopoietin 2; APC, antigen-presenting cells; αPD-1, anti-
programmed cell death protein 1; αPD-L1, anti-programmed death ligand-1; Arg1, arginase 1; β2M, beta-2 microglobulin; CCL, C-C-motif chemokine ligand; CI,
confidence interval; CSF1, macrophage colony-stimulating factor 1; CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T-lymphocyte antigen 4; CXCL12, C-X-C-motif
chemokine ligand 12; DC, dendritic cell; DCR, disease control rate; EC, endothelial cell; ESMO, European Society of Medical Oncology; FASL, Fas ligand; GM-CSF,
granulocyte–macrophage colony-stimulating factor; HLA, human leukocyte antigen; IDO, indolaimine-2, 3-deoxygenase; IFN, interferon; IL, interleukin; JAK, Janus
kinase; LAG3, lymphocyte activation gene 3; M2 Mɸ, type II macrophage; MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; NF-κB,
nuclear factor kappa B; NSCLC, non-small cell lung cancer; ORR, objective response rate; OS, overall survival; PD-1, programmed cell death protein 1; PD-L1,
programmed death ligand-1; PD-L2, programmed death ligand-2; PFS, progression-free survival; PGE2, prostaglandin E2; PS, performance status; RECIST, Response
Evaluation Criteria in Solid Tumors; STAT, signal transducer and activator of transcription; TAM, tumor-associated macrophage; TCR, T-cell receptor; TEM, effector
memory T cell; TGFβ, tumor growth factor beta; TGR, tumor growth rate; TIM3, T-cell immunoglobulin and mucin domain-3; TME, tumor microenvironment; TNF,
tumor necrosis factor; Treg, regulatory T cell; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VISTA, V-domain
immunoglobulin suppressor of T-cell activation Corresponding author at: Royal Marsden Hospital NHS Founda orresponding author at: Royal Marsden Hospital NHS Foundation Trust, 203 Fulham Road, Chelsea, London, SW3 6JJ, UK. -mail address: Sanjay.Popat@rmh.nhs.uk (S. Popat). 1. Introduction ratio for OS, 0.69), KEYNOTE-189 (combination pembrolizumab plus
chemotherapy: hazard ratio for OS, 0.59), and KEYNOTE-407 (pem-
brolizumab plus chemotherapy, squamous NSCLC: hazard ratio for OS,
0.64) [7,9,10]. Meanwhile, a growing body of evidence supports the
addition of immunotherapy to chemotherapy for patients with newly
diagnosed, metastatic NSCLC regardless of PD-L1 status (as shown by
the OS results in KEYNOTE-189 and -407, in patients with PD-L1 ex-
pression 1–49 % or PD-L1 < 1 %) [2,7,10]. Despite the advent of public health initiatives in recent years, North
America and Central and Eastern Europe continue to record some of the
highest lung cancer incidence rates globally [1]. Non-small cell lung
cancer (NSCLC) accounts for an estimated 80–90 % of all lung cancers,
with adenocarcinomas representing an increasing healthcare priority
across the USA and Europe [2]. The therapeutic landscape for stage IV NSCLC has been redefined in
recent years with the advent of targeted agents directed at specific
driver mutations and by the emergence of immunotherapies [2,3]. To
reflect the rapidly expanding therapeutic armamentarium, national and
international organizations, including the European Society of Medical
Oncology (ESMO) and the National Cancer Comprehensive Network,
have updated their recommendations to include immunotherapy stra-
tegies alongside traditional chemotherapy as front-line options (either
as monotherapies in biomarker-selected patients, or in combination
with chemotherapy) in eligible patients with advanced NSCLC without
oncogenic driver mutations [2,3]. In patients who progress after chemotherapy ± immunotherapy,
treatment with an anti-angiogenic plus docetaxel may be an option:
either nintedanib (a small molecule drug targeting the tyrosine kinases
vascular endothelial growth factor receptor [VEGFR] 1–3, platelet-de-
rived growth factor receptor α and β, and fibroblast growth factor re-
ceptor 1–3) in patients with adenocarcinoma NSCLC, or ramucirumab
(an anti-VEGFR2 antibody) in patients with performance status (PS)
0–2 [2]. However, the clinical utility of these second-line options has largely
been inferred from trials conducted in the pre-immunotherapy era. For
example, in the LUME-Lung 1 trial, patients with adenocarcinoma
NSCLC who received nintedanib plus docetaxel after failure on first-line
chemotherapy achieved a median OS of 12.6 months [11]. The REVEL
trial of post-chemotherapy ramucirumab plus docetaxel in patients with
NSCLC demonstrated a median OS of 10.5 months [12]. In the second-
line, post-chemo-immunotherapy setting, there is a lack of high-quality
evidence from prospective clinical trials to optimize second-line treat-
ment selection. 2. Emerging clinical algorithms In recent years, the development of agents targeting a wide range of
oncogenic alterations (including EGFR and BRAF driver mutations, or
gene rearrangements in ALK and ROS-1) has resulted in a clear strategy
for treatment stratification according to the molecular profile of the
tumor [2,4,5]. The emergence of immunotherapy has subsequently
revolutionized therapeutic approaches for patients with NSCLC lacking
such driver mutations [2], and has also shown potential in patients
harboring oncogenic driver mutations once molecularly targeted op-
tions have been exhausted. As the interactions between the immune system and cancer cells are
continuous and evolve throughout periods of treatment [13], an im-
portant consideration when profiling patients progressing on combi-
nation chemo-immunotherapy is the nature of their initial response,
and their pattern of progression (e.g. hyperprogression, pseudopro-
gression, or oligoprogression). Drawing on the results of published immunotherapy trials, it is
possible to categorize patients according to their response as: (i) ‘re-
sponders’ – individuals who initially respond and continue to experi-
ence clinical benefit; (ii) individuals with ‘primary innate resistance’
who appear to have no response and no benefit from treatment; and (iii)
individuals with ‘acquired resistance’, who initially respond and derive
benefit, but later acquire resistance and go on to relapse and progress
[14]. Checkpoint inhibitors targeting programmed cell death protein 1
(PD-1) and programmed death ligand-1 (PD-L1) have demonstrated
durable disease response and prolonged survival in metastatic NSCLC. For example, patients with advanced, non-squamous NSCLC without
sensitizing EGFR or ALK mutations treated with first-line pem-
brolizumab and chemotherapy achieved a response rate of 47.6 %, a
median duration of response of 11.2 months, and a median overall
survival (OS) of 22.0 months (hazard ratio 0.56; p < 0.00001 versus
chemotherapy alone) [6,7]. Other PD-1- and PD-L1-targeted agents are
now recommended by guideline bodies as first-line standard-of-care
options alongside chemotherapy [2,3]. Yet this apparently straightforward classification system obscures
the complexity of the clinical reality. Response can differ spatially
(between lesions) and also temporally – presenting immediately, ra-
pidly, or as more slowly acquired resistance [14–17]. Moreover, the
dynamic and constantly evolving nature of the immune response at the
individual patient level can be influenced by environmental and genetic
factors and/or exposure to treatment, resulting in a wide range of
possible barriers to therapeutic efficacy and a range of different re-
sponse–progression profiles, each with particular implications for sub-
sequent treatment [13]. 1. Introduction Further data on the impact of front-line chemo-im-
munotherapy
and its implications for treatment
sequencing
are
awaited. In the meantime, clinicians must take a pragmatic approach to
sequential therapy selection, guided by determinants of potential
treatment response as discussed below. However, the speed at which immunotherapies have re-shaped the
treatment landscape in this setting has resulted in a dearth of mature
clinical data to guide treatment decisions for patients who progress on
chemo-immunotherapy [2]. To facilitate such treatment decisions, we
review a range of scenarios observed in this situation, outline me-
chanistic and clinical considerations that may influence subsequent
treatment selection, and consider the potential role of anti-angiogenic
agents within this rapidly changing therapeutic landscape. https://doi.org/10.1016/j.lungcan.2020.04.009 Received 12 January 2020; Received in revised form 1 April 2020; Accepted 9 April 2020
0169-5002/ ©
2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecom
m
ons.org/licenses/BY/4.0/). Lung Cancer 144 (2020) 76–84 S. Popat, et al. 2.1. Patterns of clinical progression on immunotherapy Despite the improved outcomes offered by immunotherapy as
compared with chemotherapy alone, approximately half of patients
with advanced NSCLC do not respond, and ultimately, almost all pa-
tients relapse. Although response is not the only marker for long-term
treatment benefit with immunotherapy [8], disease progression often
drives re-evaluation of the treatment plan. At the point of progression,
many patients are eligible for additional systemic therapies, creating a
growing need for evidence to guide sequential treatment options. For instance, hyperprogression is the paradoxical occurrence of
rapid clinical and radiographic deterioration that may occur shortly
after initiating treatment. Definitions of hyperprogression vary within
the literature, with some classifying tumor growth rate (TGR) according
to tumor diameter, others by tumor volume, and some according to
Response Evaluation Criteria in Solid Tumors (RECIST) evidence of
progression within 2 months. In the context of NSCLC, hyperprogres-
sion is generally defined as a ≥2-fold increase in TGR in patients with
disease progression between baseline and first assessment by RECIST
criteria at 8 weeks. Although hyperprogression is well documented and
is not necessarily specific to immunotherapy approaches, its etiology
remains unclear. Possible explanations include oncogenic signaling For patients with non-oncogene addicted NSCLC and PD-L1 ex-
pression ≥50 %, the 2019 ESMO guidelines recommend pem-
brolizumab monotherapy [2]; although it should be noted that there is
some uncertainty regarding the decision to add chemotherapy based on
the results from KEYNOTE-042 (pembrolizumab monotherapy: hazard 77 S. Popat, et al. Lung Cancer 144 (2020) 76–84 activation, upregulation of alternative immune checkpoints, or mod-
ulation of other protumor immune subsets. Predictive studies have
failed to show any association between hyperprogressive disease and a
range of disease characteristics (e.g. tumor burden, histologic subtype,
number of metastatic sites) or therapeutic approaches (previous lines of
chemotherapy or prior treatment), but age appears to be a risk factor. In
one study, almost 20 % of patients older than 65 years developed hy-
perprogressive disease compared with only 5 % of those aged 65 years
or younger (p = 0.018) [16,17]. certain types of macrophages and tolerogenic dendritic cells. Under
physiological conditions, PD-1, PD-L1, and PD-L2 play important roles
in regulating T-cell activation during peripheral tolerance: macro-
phages and tolerogenic dendritic cells prevent autoimmunity by in-
hibiting autoreactive T cells that may cause tissue damage [19]. 2.1. Patterns of clinical progression on immunotherapy Acti-
vation of the PD-1/PD-L1 pathway can result in the induction of T cell-
mediated anergy, apoptosis, and ‘exhaustion’, initiating T-cell sup-
pression. Solid tumors can ‘hijack’ the PD-1/PD-L1 axis, causing over-
expression of PD-L1 and inducing immune suppression and evasion,
thus inhibiting the attack of conventional cytotoxic CD8 + T cells and
preventing tumor lysis. Inhibition of the PD-1/PD-L1 pathway, there-
fore, triggers tumor antigen recognition, proliferation, infiltration, and
activation of cytotoxic CD8 + T cells, resulting in an antitumor immune
response [19]. Another rare progression–response profile is that of pseudopro-
gression, estimated to occur in approximately 2–6 % of patients treated
with immunotherapy [11]. Pseudoprogression is characterized by a
transient enlargement of the tumor (or metastatic sites) before regres-
sion in size, and is attributed to an initial inflammatory reaction that
may resemble a tumor flare [16]. Immuno-histochemical evaluations
suggest baseline tumor cells may increase in number in parallel with an
inflammatory response consisting of activated cytotoxic lymphocytes
(CD8 + T cells), TIA-1 (an apoptosis-promoting protein) and granzyme
B (a protein necessary for the induction of apoptosis by cytotoxic T
cells). In light of its transient nature, in order to differentiate pseudo-
progression from true progression and necessary clinical action, new
radiographic assessment protocols have been developed that mandate
specific time intervals and application of an immunotherapy-specific
RECIST classification system before evaluation of response to im-
munotherapy [16,17]. Successful antitumor immune response following immunotherapy
with PD-1/PD-L1 immune checkpoint inhibitors, therefore, requires the
reactivation and clonal proliferation of antigen-experienced T cells
present in the tumor microenvironment (TME) [13,14]. Successful
processing, presentation, and recognition of tumor-associated peptide
antigens is required to generate tumor-specific CD8 + T cells with tu-
moricidal potential. The first signal for T-cell activation occurs when a
unique T-cell receptor recognizes a major histocompatibility complex
(MHC)-bound tumor antigen. Full T-cell activation is then triggered by
the binding of the co-stimulatory CD28 receptor on T cells by B7 on the
antigen-presenting cells. The resultant tumor-specific CD8 + T cells
subsequently differentiate into effector T cells before undergoing clonal
expansion and trafficking to the TME, where they kill tumor cells dis-
playing the tumor-associated antigen on human leukocyte antigen
(HLA). It should be noted that hyperprogression and pseudoprogression are
rare events in patients who receive first-line chemo-immunotherapy;
they are seen mainly in patients treated with pembrolizumab mono-
therapy. 3.1. Insufficient generation of antitumor T cells Successful immunotherapy with immune checkpoint inhibitors re-
quires the reactivation of T cells directed at tumor-specific mutant
proteins. Conversely, a lack of suitable neoantigens and alterations in
antigen processing (and/or presentation) is associated with impaired
antitumor immune response. Tumor-intrinsic mechanisms of immune evasion, therefore, include
genetic and epigenetic alterations that influence neoantigen formation,
presentation, and/or processing. They also include alterations in cel-
lular signaling pathways that disrupt the action of cytotoxic T cells. Alterations in genes encoding components of the antigen processing
and/or presentation apparatus (e.g. class I MHC, beta-2 microglobulin
[β2M]) can also lead to resistance to immune checkpoint inhibitor
therapy. Furthermore, downregulation of HLA class I molecules and
loss of β2M expression may also be implicated: loss of β2M expression
results in impaired cell-surface expression of MHC class I, which in turn
impairs antigen presentation to cytotoxic T cells [14,20]. 3. Mechanisms of resistance to immunotherapy In the absence of robust data to delineate the effect of approved
immunotherapies on emergent tumor biology, the mechanisms under-
pinning different patterns of response–progression could potentially
guide subsequent treatment selection in order to optimize outcomes for
individual patients. Licensed PD-1/PD-L1 immune checkpoint inhibitors function by
preventing one mechanism of immune evasion available to the tumor. The immune checkpoint molecule PD-1 is expressed on the extracellular
surface of natural killer T cells, B cells, dendritic cells, monocytes/
macrophages, and CD4+ and CD8 + T cells. PD-L1 is present on im-
mune cells, as well as on certain types of cancer and stromal cells. Interaction of PD-1 with PD-L1 inhibits the activation, proliferation,
and survival of T cells [19]. The PD-1 receptor also interacts with
programmed death ligand-2 (PD-L2), which is selectively expressed on 2.1. Patterns of clinical progression on immunotherapy In cases of hyperprogression on pembrolizumab monotherapy,
treatment with platinum-based doublet chemotherapy is likely; treat-
ment with a bevacizumab-containing doublet could be considered,
given the potential immunomodulatory role of bevacizumab, but as yet,
there is no clinical data to robustly support this strategy given the rarity
of hyperprogression. For long-term immunologic memory, a subset of effector T cells
must differentiate into effector memory T (TEM) cells; these are main-
tained for life and respond to antigen re-challenge [14]. Disease pro-
gression (or ‘failure’ of immune checkpoint blockade), can result from
defects in any of the steps that underpin this process, i.e. from in-
sufficient generation of antitumor T cells, inadequate tumor-specific T-
cell function, or impaired formation of T-cell memory [14]. Mechanistic
defects that impair the effect of PD-1 blockade can arise from a number
of tumor-intrinsic or -extrinsic factors, presenting either at the time of
initial immunotherapy trial (primary/innate resistance) or after a
period of initial response, resulting in progression (acquired resistance)
(Fig. 1) [13,14]. A third form of progression has been identified as oligoprogression. Oligometastatic disease is an intermediate state between localized and
widespread metastatic cancer, often defined in NSCLC as fewer than
five sites of disease. The term usually describes patients with synchro-
nous or metachronous metastatic disease at presentation, but recent
genomic studies have revealed distinct clonal evolution at each site of
metastatic disease, leading to the hypothesis that individual sites may
develop treatment resistance or increased metastatic potential in-
dependent of the primary site of disease or even other metastatic sites
[15]. This phenomenon is particularly associated with oncogene-ad-
dicted
NSCLC. Results
from
the
phase
II
SABR-COMET
trial
(NCT01446744) indicate that patients with oligoprogression limited to
only a few sites of disease may warrant treatment with locally ablative
therapies [18], and further data are awaited from trials such as NRG-
LU-002 (NCT03137771). 3.2. Inadequate tumor-specific T-cell function APC, antigen-presenting cells; αPD-1, anti-programmed cell death protein 1; αPD-L1, anti-programmed death ligand-1; Arg1, arginase 1; β2M, beta-2 m
CTLA-4, cytotoxic T-lymphocyte antigen 4; IDO, indolaimine-2, 3-deoxygenase; IFN, interferon; LAG3, lymphocyte activation gene 3; M2 Mɸ, type II
MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; PD-L1, programmed death li anel: basic steps involved in generation of tumor-specific T cells, effector T-cell function, and formation of memory T cells. anel: putative mechanisms of innate and/or acquired resistance to immune checkpoint inhibitor therapy. Lower panel: putative mechanisms of innate and/or acquired resistance to immune checkpoint inhibitor therapy. APC, antigen-presenting cells; αPD-1, anti-programmed cell death protein 1; αPD-L1, anti-programmed death ligand-1; Arg1, arginase 1; β2M, beta-2 microglobulin;
CTLA-4, cytotoxic T-lymphocyte antigen 4; IDO, indolaimine-2, 3-deoxygenase; IFN, interferon; LAG3, lymphocyte activation gene 3; M2 Mɸ, type II macrophage;
MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand-1; PGE2,
prostaglandin E2; TCR, T-cell receptor; TIM3, T-cell immunoglobulin and mucin domain-3; Treg, regulatory T cell; VISTA, V-domain immunoglobulin suppressor of T-
cell activation. L1-independent mechanisms of immune escape, development of re-
sistant mutations, T-cell exhaustion, and the development of an im-
munosuppressive TME. effects of interferon gamma, and analysis of tumors in patients who did
not respond to anti-CTLA-4 therapy has revealed an enriched frequency
of mutations in the interferon gamma pathway genes, interferon
gamma receptor 1 and 2, JAK2, and interferon regulatory factor 1. Mutations in any of these genes resulting in inhibition of interferon
gamma-related signaling could, therefore, result in primary resistance
to anti-CTLA-4 therapy and lack of PD-L1 expression upon interferon
gamma exposure [13]. For instance, a number of PD-L1-independent mechanisms of im-
mune escape have been identified, such as immune suppressive cyto-
kines, immune inhibitory metabolites, immune suppressive cells, and
the over-expression of alternate immune checkpoints or co-inhibitory
receptors (e.g. cytotoxic T-lymphocyte antigen 4 [CTLA-4], lymphocyte
activation gene 3 [LAG3], T-cell immunoglobulin and mucin domain-3
[TIM3], and V-domain immunoglobulin-containing suppressor of T-cell
activation [VISTA]) [13,14,21]. Targeting PD-1/PD-L1 in isolation,
therefore, does not necessarily silence tumor pathogenesis mediated by
one or a combination of these alternative pathways. Heterogeneity in PD-1+ and CD8+ populations also appears to be
associated with differential response to PD-1 immune checkpoint in-
hibitor therapy and functional exhaustion of T cells. 3.2. Inadequate tumor-specific T-cell function Indeed, partial
exhaustion of PD-1+, CTLA-4+, and CD8+ infiltrating T cells has been
shown to correlate with PD-1 response, and exhausted PD-1+ and CD8
+ T cells display a distinct chromatin landscape compared with effector
T cells and TEM cells. These epigenetically distinct T cells appear to
influence whether or not exhausted PD-1+ T cells can be repro-
grammed to avoid terminal exhaustion and dysfunction [14]. Resistance mutations can also arise that inhibit the clinical efficacy
of ongoing immunotherapy. Whole exome sequencing of tumors from
patients who developed resistance following initial clinical response to
PD-1 blockade suggests that mutations in Janus kinases (JAK) 1 and 2
may be implicated [14]. Downregulating or mutating molecules in-
volved in the interferon gamma signaling pathway (which goes through
the interferon gamma receptor chains JAK1 and/or JAK2 and the signal
transducer and activators of transcription [STATs]) could enable eva-
sion of the effects of interferon gamma [13]. Preclinical data support
the hypothesis that mutations or epigenetic silencing of molecules in
the interferon receptor signaling pathway result in loss of the antitumor 3.2. Inadequate tumor-specific T-cell function A range of tumor-intrinsic and -extrinsic factors can also contribute
to inadequate tumor-specific T-cell function and diminished clinical
effect of PD-1/PD-L1 immune checkpoint inhibitors. These include PD- 78 S. Popat, et al. Lung Cancer 144 (2020) 76–84 Fig. 1. Response and resistance to immune checkpoint inhibitor therapy. Adapted from Jenkins et al. 2018 [14]. Upper panel: basic steps involved in generation of tumor-specific T cells, effector T-cell function, and formation of memory T cells. Lower panel: putative mechanisms of innate and/or acquired resistance to immune checkpoint inhibitor therapy. APC, antigen-presenting cells; αPD-1, anti-programmed cell death protein 1; αPD-L1, anti-programmed death ligand-1; Arg1, arginase 1; β2M, beta-2 microglobul
CTLA-4, cytotoxic T-lymphocyte antigen 4; IDO, indolaimine-2, 3-deoxygenase; IFN, interferon; LAG3, lymphocyte activation gene 3; M2 Mɸ, type II macrophag
MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand-1; PGE
prostaglandin E2; TCR, T-cell receptor; TIM3, T-cell immunoglobulin and mucin domain-3; Treg, regulatory T cell; VISTA, V-domain immunoglobulin suppressor of
cell activation. S. Popat, et al. Lung Cancer 144 (2020) 76– Fig. 1. Response and resistance to immune checkpoint inhibitor therapy. Adapted from Jenkins et al. 2018 [14]. Upper panel: basic steps involved in generation of tumor-specific T cells, effector T-cell function, and formation of memory T cells. Lower panel: putative mechanisms of innate and/or acquired resistance to immune checkpoint inhibitor therapy. APC, antigen-presenting cells; αPD-1, anti-programmed cell death protein 1; αPD-L1, anti-programmed death ligand-1; Arg1, arginase 1; β2M, beta-2 microglobulin
CTLA-4, cytotoxic T-lymphocyte antigen 4; IDO, indolaimine-2, 3-deoxygenase; IFN, interferon; LAG3, lymphocyte activation gene 3; M2 Mɸ, type II macrophage
MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand-1; PGE2
prostaglandin E2; TCR, T-cell receptor; TIM3, T-cell immunoglobulin and mucin domain-3; Treg, regulatory T cell; VISTA, V-domain immunoglobulin suppressor of T
cell activation. S. Popat, et al. Lung Cancer 144 (2020) 7684 Fig. 1. Response and resistance to immune checkpoint inhibitor therapy. Adapted from Jenkins et al. 2018 [14]. Fig. 1. Response and resistance to immune checkpoint inhibitor therapy. Adapted from Jenkins et al. 2018 [14]. Upper panel: basic steps involved in generation of tumor-specific T cells, effector T-cell function, and formation of memory T cells. Lower panel: putative mechanisms of innate and/or acquired resistance to immune checkpoint inhibitor therapy. 3.4. Immunosuppression within the TME VEGF also directly inhibits dendritic cell maturation and activation
of antigen-specific Tregs and reduces immune cell–EC interactions in
angiogenic vessels. Furthermore, there is a growing list of hemato-
poietic cell types (e.g. tumor-associated macrophages, MDSCs, TIE2+
monocytes, immature dendritic cells, and Tregs) that, when appro-
priately polarized, can promote both immunosuppression and angio-
genesis through production of VEGF and other factors, such as basic
fibroblast growth factor, chemokine (C-C-motif) ligand 2 and ANG2
[32]. Experimental studies have shown that depletion of these im-
munosuppressive cell types can enhance antitumor immune responses
and has the potential to overcome innate resistance [14]. The TME is also growing in recognition as another driver of ac-
quired resistance to treatment with PD-1/PD-L1 immune checkpoint
inhibitors, as immunosuppression within the TME has been shown to
impair the efficacy of PD-L1 therapy [14,21]. An immunosuppressive TME is characterized by high levels of im-
mune-suppressing cytokines and/or metabolites; by the recruitment of
immune
suppressive
cells
(e.g. myeloid-derived
suppressor
cells
[MDSCs], and regulatory T cells [Tregs]); by mutations in key effector
pathways; and by high levels of PD-L1 expression [14,21]. Preclinical
models have shown an association between elevation of immune-sup-
pressive cell types (including Tregs, MDSCs, Th2 CD4 + T cells, and M2-
polarised tumor-associated macrophages) in the TME and diminished
immune checkpoint inhibitor efficacy. These cell types promote an
immunosuppressive TME that inhibits antitumor cytotoxic and Th1-
directed T-cell activities, primarily through the release of cytokines,
chemokines, and other soluble mediators [14]. The discovery that VEGF was a key mediator of angiogenesis
marked it out as a key therapeutic target, and led to the development of
a number of agents with the potential to induce regression of angio-
genic vessels and starve tumors of their blood supply and nutrients
[33]. There are currently two anti-angiogenic agents licensed for use in
metastatic NSCLC without oncogenic driver mutations in combination
with docetaxel following progression on chemotherapy: nintedanib and
ramucirumab [30,31]. Mutations associated with development of an immunosuppressive
or ‘cold’ TME include STK11/LKB1 and KEAP1 (often found in con-
junction with RAS mutations in NSCLC), which may result in primary
resistance to immunotherapy [22–24]. 3.4. Immunosuppression within the TME For example, in the phase III
MYSTIC study of durvalumab (anti-PD-L1) and tremelimumab (anti-
CTLA-4), STK11/LKB1 and KEAP1 mutations were present in 16 % and
18 % of evaluable patients, respectively, and both were associated with
poorer OS than in patients with wild-type variants; although there are
currently no data to suggest STK11/LKB1 or KEAP1 mutations are
predictive of a poorer response to immunotherapy compared with
chemotherapy [25]. A retrospective analysis of patients with non-
squamous NSCLC treated with first-line chemotherapy with or without
pembrolizumab has shown that STK11/LKB1 alterations define a sub-
group of patients with inferior clinical outcomes and a lack of clinical
benefit from addition of pembrolizumab [26]. The predictive value of
STK11/LKB1 and KEAP1 mutations remains to be confirmed in a pro-
spective clinical trial; current data suggest a strong prognostic poten-
tial, regardless of treatment [25]. In addition to its ability to suppress sprouting angiogenesis and
delay tumor growth, anti-angiogenic activity can transiently normalize
tumor vasculature and offer complementary immunomodulatory ef-
fects. Normalization of the tumor vasculature can improve blood per-
fusion and oxygenation, thereby enabling increased infiltration of im-
mune
effector
cells
and
converting
an
immunosuppressive
microenvironment to an immunosupportive environment (Fig. 2)
[33–35]. This hypothesis is supported by preclinical evidence of an ‘angio-
immunogenic switch’ mechanism whereby anti-angiogenics create a
transient window for immune system detection and infiltration of an-
ticancer therapies into the TME. In a model of immune-tolerant breast
cancer, lower-doses (20 mg/kg or 10 mg/kg) of an anti-VEGFR2 anti-
body (DC101) have been shown to result in a more homogeneous dis-
tribution of functional blood vessels and improved tissue perfusion than
full-dose DC101 (40 mg/kg). The lower-dose also enhanced the antic-
ancer efficacy of vaccine therapy, reducing tumor volume and sig-
nificantly slowing tumor growth compared with full-dose DC101. The
lower-dose DC101 regimen reverted the immunosuppressive TME to an
immunosupportive environment, with T-cell tumor infiltration in-
versely correlated with the DC101 dose [36]. 3.3. Impaired formation of T-cell memory While there is compelling evidence to support the ability of PD-1/
PD-L1 immune checkpoint inhibitors to reinvigorate cytotoxic T lym-
phocytes and achieve long-term clinical benefit in some patients, this
appears to be contingent upon effective TEM formation. If TEM formation 79 S. Popat, et al. Lung Cancer 144 (2020) 76–84 is impaired in any way, an initial clinical response may dissipate over
time, leading to acquired resistance and potential disease progression. Expansion of the intratumoral TEM compartment has been demon-
strated in response to PD-1 blockade, and to be positively associated
with therapeutic response. Ongoing research aims to elucidate the
mechanisms underlying TEM expansion following PD-1 blockade, but
distinct transcriptional programs associated with naïve, acute effector,
memory, and exhausted T-cell states have been identified; there is
emerging evidence that T-cell exhaustion is associated with epigenetic
changes that appear to limit the durability of CD8 + T-cell function
following PD-1 blockade [14]. is impaired in any way, an initial clinical response may dissipate over
time, leading to acquired resistance and potential disease progression. abnormalities are a hallmark of many solid tumors and are known to
facilitate immune evasion. The abnormalities stem from elevated levels
of proangiogenic factors, such as vascular endothelial growth factor
(VEGF) and angiopoietin 2 (ANG2). In addition to regulating angio-
genesis, VEGF also plays a role in immunosuppression as abnormal
vessels and impaired perfusion can restrict entry of cytotoxic drugs and
immune cells from the circulation into tumors. To infiltrate the tumor
and integrate into the TME, immune cells must enter the tumor blood
vessels, adhere to the endothelium, and transmigrate across the vessel
wall. The presence of angiogenic molecules such as VEGF within the
TME can control the trafficking of immune cells to the tumor by altering
the expression of adhesion molecules on endothelial cells (ECs) and
immune cells [33]. Expansion of the intratumoral TEM compartment has been demon-
strated in response to PD-1 blockade, and to be positively associated
with therapeutic response. Ongoing research aims to elucidate the
mechanisms underlying TEM expansion following PD-1 blockade, but
distinct transcriptional programs associated with naïve, acute effector,
memory, and exhausted T-cell states have been identified; there is
emerging evidence that T-cell exhaustion is associated with epigenetic
changes that appear to limit the durability of CD8 + T-cell function
following PD-1 blockade [14]. 4. Targeting immunotherapy resistance using anti-angiogenics In addition, tumor-infiltrating CTLs ty-
pically upregulate PD-1, marking them as
dysfunctional or ‘exhausted’ and limiting their
cytotoxic
potential
against
tumor
cells. Overall, the consequence of aberrant tumor
angiogenesis and vascular abnormality is an
immunosuppressive TME. ANG2, angiopoietin 2; CCL, C-C-motif chemo-
kine ligand; CXCL12, C-X-C-motif chemokine
ligand 12; CSF1, macrophage colony-stimu-
lating factor 1; CTL, cytotoxic T lymphocyte;
EC, endothelial cell; DC, dendritic cell; FASL,
Fas ligand; GM-CSF, granulocyte–macrophage
colony-stimulating factor; IL, interleukin; PD-
, tumor microenvironment; TGFβ, transforming 1, programmed cell death protein 1; PD-L1, programmed death ligand-1; TAM, tumor-associated macrophage; TME
growth factor beta; Treg, regulatory T cell; VEGF, vascular endothelial growth factor. Although small, these retrospective analyses provide initial evi-
dence, in a real-world setting, demonstrating consistent clinical benefit
with third-line anti-angiogenic therapies (in combination with doc-
etaxel) after failure on a checkpoint inhibitor. Thus, the rational se-
quencing of anti-angiogenics after failure on immunotherapy may be a
promising approach that warrants further investigation in future clin-
ical trials. without chemotherapy). Prospective data will be generated by trials
such as the planned trial of the combination of nab-paclitaxel and
nintedanib or nab-paclitaxel and placebo in relapsed NSCLC adeno-
carcinoma (NCT03361319). In the meantime, however, published data
are available from two real-world evaluations of nintedanib plus doc-
etaxel following progression after chemotherapy followed by PD-1
blockade [38,39]. without chemotherapy). Prospective data will be generated by trials
such as the planned trial of the combination of nab-paclitaxel and
nintedanib or nab-paclitaxel and placebo in relapsed NSCLC adeno-
carcinoma (NCT03361319). In the meantime, however, published data
are available from two real-world evaluations of nintedanib plus doc-
etaxel following progression after chemotherapy followed by PD-1
blockade [38,39]. The first study involved a retrospective analysis of centers partici-
pating in the Spanish nintedanib named patient use program. Eligible
patients (n = 11) received nintedanib plus docetaxel after progression
on chemotherapy and immune checkpoint inhibitor therapy. Third-line
treatment with nintedanib plus docetaxel was associated with an ob-
jective response rate (ORR) of 36 %, a disease control rate (DCR) of 82
%, and a median progression-free survival (PFS) of 3.2 months [38]. Anti-angiogenics may also be combined with immunotherapy and
chemotherapy. Recent data from the phase III IMPOWER150 trial offer
proof of concept of the clinical relevance of the interplay between an-
giogenesis and immunosuppression. 4. Targeting immunotherapy resistance using anti-angiogenics Considering progression on immunotherapy from a mechanistic
perspective can help to identify therapeutic targets with the potential to
delay or reverse resistance and prolong therapeutic effect. Among the
most promising strategies under investigation are those targeting mul-
tiple mechanisms of immune escape using dual immune checkpoint
inhibitor blockade (e.g. targeting both CTLA-4 and PD-1), and those
using radiation (particularly stereotactic body radiation therapy) to
prime
immunity
and
enhance
the
effect
of
immunotherapy
[13,14,27–29]. However, the approach with one of the strongest me-
chanistic foundations, and with immediate clinical relevance given the
availability of licensed second-line agents, is the use of anti-angio-
genics. Mechanistically, Wei et al. further demonstrate that the local im-
mune landscape and PD-L1 heterogeneity may give rise to differing
cancer severity hallmarks and clinical outcomes, predisposing some
tumors to particular angiogenic and treatment response signatures. The
authors report that nuclear factor kappa B signals elicited by macro-
phage inflammatory responses generate PD-L1+ cancer cells with ag-
gressive survival capabilities, which support angiogenesis and have the
ability to metastasize. Meanwhile, STAT1 signals triggered by activated
T cells generate PD-L1+ cancer cells susceptive to apoptosis (Fig. 3)
[37]. There is an increasing body of preclinical and clinical evidence to
suggest that sustained angiogenesis and immunosuppression are inter-
connected
processes
with
shared
regulators
[30–32]. Vascular At the time of writing, there are no published trial data from pro-
spective, randomized controlled trials evaluating the role of anti-an-
giogenic therapy following progression on immunotherapy (with/ 80 Lung Cancer 144 (2020) 76–84 S. Popat, et al. 1, programmed cell death protein 1; PD-L1, programmed death ligand-1; TAM, tumor-associated macrophage; TME
growth factor beta; Treg, regulatory T cell; VEGF, vascular endothelial growth factor. Abnormalities in the tumor vasculature result
in hypoxia and acidosis of the TME, which in
turn
contribute
to
immunosuppression
via
several mechanisms. These mechanisms in-
clude: increased accumulation, activation, and
expansion
of immunosuppressive
Tregs;
re-
cruitment of inflammatory monocytes and
TAMs; suppression of DC maturation, which
results in impaired antigen presentation and
activation of tumor-specific CTLs; and expan-
sion of abnormal ECs with immunosuppressive
phenotypes. Importantly,
the
PD-1/PD-L1
pathway is often activated in the TME as a
mechanism to evade anticancer immune re-
sponses, with upregulation of PD-L1 expression
on TAMs, DCs, and ECs, as well as on tumor
cells. 4. Targeting immunotherapy resistance using anti-angiogenics Lung Cancer 144 (2020) 76–84 Fig. 3. Role of the local immune landscape on tumor PD-L1 heterogeneity and sensitivity to therapy. Adapted from Wei et al. 2019 [37]. NF-κB signals elicited by macrophage inflammatory responses generate PD-L1+ cancer cells with survival, angiogenic, and metastatic capabilities. Meanw
signals triggered by activated T cells can induce susceptibility to apoptosis in PD-L1+ cancer cells. Ab, antibody; IFN, interferon; IL, interleukin; NF-κB, nuclear factor kappa B; PD-L1, programmed death ligand-1; STAT, signal transducer and activat
scription; TNF, tumor necrosis factor. S. Popat, et al. Lung Cancer 144 (2 Fig. 3. Role of the local immune landscape on tumor PD-L1 heterogeneity and sensitivity to therapy. Adapted from Wei et al. 2019 [37]. p
NF-κB signals elicited by macrophage inflammatory responses generate PD-L1+ cancer cells with survival, angiogenic, and metastatic capabilities. Meanwhile STAT1
signals triggered by activated T cells can induce susceptibility to apoptosis in PD-L1+ cancer cells. Ab, antibody; IFN, interferon; IL, interleukin; NF-κB, nuclear factor kappa B; PD-L1, programmed death ligand-1; STAT, signal transducer and activator of tran-
scription; TNF, tumor necrosis factor. and comorbidities, and in populations more representative of real-
world case-loads. Of interest within this context is the open-label, phase
IIb SENECA trial of nintedanib plus 3-weekly or weekly schedules of
docetaxel. The weekly docetaxel schedule (33 mg/m2 on days 1 and 8
of each 21-day cycle) was better tolerated than the 3-weekly schedule
(75 mg/m2), with no statistically significant difference in efficacy [45]. Furthermore, balancing health and quality of life outcomes with af-
fordability will be non-trivial in light of the high economic impact that
immunotherapies present to healthcare systems [46]. inhibitor, either alone or in combination (NCT03689855); and a trial of
ramucirumab with nivolumab in patients who progressed after im-
munotherapy (either alone or in combination; the trial will also in-
vestigate immunotherapy-naïve patients; NCT03527108). Irrespective of their future position within advanced NSCLC treat-
ment protocols, optimizing the clinical benefit of anti-angiogenics will
require consideration of the apparent dose- and time-dependent nature
of their immunomodulatory effects [19,35]. Central to achieving research efficiencies and the smooth translation
of emerging knowledge into effective clinical approaches will be the
development and use of a standard taxonomy for resistance classifica-
tion and response–progression profiles. 4. Targeting immunotherapy resistance using anti-angiogenics Ideally, related studies will also
include paired re-biopsies at the time of progression, to enable better
definition of the immunological stromal landscape, determination of
biological correlates of resistance patterns, and new vulnerabilities that
may benefit from the addition of an anti-angiogenic therapy. 5. Future perspectives A recent Delphi consensus exercise conducted in Spain asked ex-
perts to consider the optimal selection of second- or later-line treat-
ments
following
progression
on
first-line
immunotherapy ±
chemotherapy for advanced adenocarcinoma; in line with current
ESMO guidelines, combined docetaxel and nintedanib was considered
to be a valid option for patients progressing on prior lines of chemo-
immunotherapy [2,44]. Nevertheless, prospective clinical data are es-
sential to ensure that management decisions for NSCLC are supported
by a robust evidence base. As the collective experience with first-line
immunotherapy for metastatic NSCLC matures, the implications of
different therapeutic approaches and their impact on tumor profile and
subsequent treatment response will become clearer, helping clinicians
navigate current therapeutic uncertainties. In parallel, targeted re-
search efforts will be required, not only to provide proof-of-concept
data for emerging therapeutic strategies, but also to facilitate evalua-
tion of their long-term risk–benefit profiles and health economic im-
plications. 4. Targeting immunotherapy resistance using anti-angiogenics The trial investigated the combi-
nation of atezolizumab (A) plus anti-angiogenic bevacizumab (B) plus
chemotherapy (C) versus AC and BC in chemotherapy-naïve patients
with metastatic non-squamous NSCLC. Addition of atezolizumab to BC
(i.e. ABC) resulted in a significant improvement in both PFS and OS,
regardless of patients’ PD-L1 expression or EGFR and ALK genetic al-
teration status. The authors proposed that the efficacy of atezolizumab
may have been enhanced by the addition of bevacizumab and its ability
to reverse VEGF-mediated immunosuppression [42]. This was particu-
larly evident for the subset of patients with EGFR-mutant NSCLC, in
which the bevacizumab-containing combination ABC resulted in an
improved OS compared with the BC control [43]. As this survival ad-
vantage was not observed in the AC investigational arm, the results
suggest a potential synergistic effect for bevacizumab and atezoli-
zumab, at least in patients with EGFR-mutant NSCLC. These findings are reinforced by the interim results from the non-
interventional VARGADO study, involving patients (n = 40) who re-
ceived nintedanib plus docetaxel following first-line chemotherapy and
second-line immune checkpoint inhibitor therapy [39,40]. At the time
of analysis (data cut-off: August 1, 2019), median duration of follow-up
was 7.1 months for patients treated with nintedanib plus docetaxel. Median PFS was 7.2 months (95 % CI: 2.9–8.7). ORR and DCR data
were available for 29 patients: partial response rate was 45 % and DCR
was 86 %. Grade ≥3 treatment-emergent adverse events occurred in 43
% of patients; serious treatment-emergent adverse events occurred in
48 % of patients; and 30 % of patients discontinued due to treatment-
emergent adverse events [40]. Ongoing or planned trials investigating anti-angiogenics in combi-
nation with immunotherapies in patients with NSCLC in a post-
(chemo)-immunotherapy setting include: a trial of nivolumab, ipili-
mumab, and nintedanib in patients who develop resistance to immune
checkpoint inhibitor therapy (as well as a separate treatment arm in
newly diagnosed patients; NCT03377023); a trial of ramucirumab with
atezolizumab in patients previously treated with an immune checkpoint There are also complementary data for ramucirumab plus docetaxel
following failure of nivolumab in metastatic NSCLC. Among patients (n
= 20) included in a published retrospective analysis, ORR was 60 %
and DCR was 90 %. Six patients had stable disease and two had pro-
gressive disease. Gastrointestinal adverse events were frequently ob-
served in almost all (n = 19/20) patients [41]. 81 S. Popat, et al. Financial and competing interests disclosure SP received personal fees from BMS, Roche, Takeda, AstraZeneca,
Pfizer, MSD, EMD Serono, Guardant Health, AbbVie, Boehringer
Ingelheim, Tesaro, OncLive and Medscape. g
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speaker’s bureau from Boehringer Ingelheim, F. Hoffmann-La Roche,
Lilly, AstraZeneca, BMS, MSD, Merck, Novartis, Pfizer and Celgene. SN received personal fees for advisor/speaker’s bureau from
AstraZeneca, Boehringer Ingelheim, BMS, Takeda, Pfizer, Roche, MSD,
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to overcoming immunotherapy resistance using existing licensed
agents. The
intertwined
regulation
of
VEGF
signaling
and
im-
munosuppression in the TME clearly supports consideration of anti-
angiogenic therapy to target immunosuppression in the TME and
trigger
an
‘angio-immunogenic
switch’
back
towards
an
im-
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Hypothalamic Glial-to-Neuronal Signaling during Puberty: Influence of Alcohol
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International journal of environmental research and public health/International journal of environmental research and public health
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Int. J. Environ. Res. Public Health 2011, 8, 2876-2894; doi:10.3390/ijerph8072894 Int. J. Environ. Res. Public Health 2011, 8, 2876-2894; doi:10.3390/ijerph8072894 International Journal of
Environmental Research and
Public Health
ISSN 1660-4601
www.mdpi.com/journal/ijerph International Journal of
Environmental Research and
Public Health
ISSN 1660-4601
www.mdpi.com/journal/ijerph Keywords: alcohol; puberty; transforming growth factor-α; glia; RPTPβ 1. Introduction The hypothalamic region of the brain plays an important role in synchronizing events leading to the
activation of the mammalian puberty. This process requires the interactive participation of both glial
and neuronal regulatory circuitries that serve to control the secretion of luteinizing hormone-releasing
hormone (LHRH) neurons [1,2]. The secretory activity of LHRH neurons is triggered by several
transsynaptic inputs of both inhibitory and excitatory nature [1,3,4]. The decreased release of
inhibitory neurotransmitters such as gamma amino butyric acid and the opiod peptides [5,6] as well as
the increased release of excitatory neurotransmitters such as excitatory amino acids [7,8], transforming
growth factor alpha (TGFα) [9], insulin-like growth factor-1 [10,11], and the kisspeptins [12] are
capable of initiating the cascade of events leading to increased LHRH secretion at puberty. The glial
cells within the medial basal hypothalamus (MBH) are key components of the central regulatory
system that facilitate prepubertal LHRH release via pathways initiated by growth factors and cell
adhesion molecules that act on LHRH neuron terminals to stimulate their secretory activity [13,14]. Growth factors of glial origin are important because they are intimately involved in glial-neuronal
signaling processes by which the glial cells, through their close association with LHRH nerve
terminals in the MBH, regulate LHRH secretion during puberty in rodents [13,15] and primates [16]. Growth factors such as basic fibroblast growth factor (bFGF) [17,18], transforming growth factor
β (TGF-β) [19] and IGF-1 [10,20] have been shown to act directly on LHRH neurons to facilitate
LHRH release. In contrast to these growth factors, members of the epidermal growth factor (EGF)
family, including EGF itself, TGFα, and the neuregulins act indirectly on LHRH release through
specific erbB receptors that have an extracellular ligand binding domain linked to a cytoplasmic
domain containing tyrosine kinase activity [14,21,22]. While all of these peptides can influence
LHRH release, a portion of this review will concentrate specifically on EGF/TGFα influences, since
they have been shown to play a pivotal role in the glial control of neuronal LHRH secretion at the time
of puberty. During the past decade, evidence has accumulated suggesting LHRH secretory activity is also
modulated by a specific glial-neuronal gene family which synthesizes adhesion/signaling proteins
involved in the functional and structural integrity of bi-directional glial-neuronal communications. Int. J. Environ. Res. Public Health 2011, 8 Int. J. Environ. Res. Public Health 2011, 8 2877 Vinod K. Srivastava, Jill K. Hiney and W. Les Dees * Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M
University, College Station, TX 77843-4458, USA; E-Mails: vsrivastava@cvm.tamu.edu (V.K.S);
jhiney@cvm.tamu.edu (J.K.H.) * Author to whom correspondence should be addressed; E-Mail: ldees@cvm.tamu.edu;
Tel.: +1-979-8451430; Fax: +1-979-847-9038. Received: 26 May 2011; in revised form: 27 June 2011 / Accepted: 12 July 2011 /
Published: 14 July 2011 Received: 26 May 2011; in revised form: 27 June 2011 / Accepted: 12 July 2011 /
Published: 14 July 2011 Abstract: Mammalian puberty requires complex interactions between glial and neuronal
regulatory systems within the hypothalamus that results in the timely increase in the
secretion of luteinizing hormone releasing hormone (LHRH). Assessing the molecules
required for the development of coordinated communication networks between glia and
LHRH neuron terminals in the basal hypothalamus, as well as identifying substances
capable of affecting cell-cell communication are important. One such pathway involves
growth factors of the epidermal growth factor (EGF) family that bind to specific erbB
receptors. Activation of this receptor results in the release of prostaglandin-E2 (PGE2) from
adjacent glial cells, which then acts on the nearby LHRH nerve terminals to elicit release
of the peptide. Another pathway involves novel genes which synthesize adhesion/signaling
proteins responsible for the structural integrity of bi-directional glial-neuronal
communication. In this review, we will discuss the influence of these glial-neuronal
communication pathways on the prepubertal LHRH secretory system, and furthermore,
discuss the actions and interactions of alcohol on these two signaling processes. Keywords: alcohol; puberty; transforming growth factor-α; glia; RPTPβ 2. EGF/TGFα Family of Growth Factors and Puberty Growth factors of glial cell origin, acting via receptor tyrosine kinases, have been shown to be key
components of the mechanism by which hypothalamic glial cells regulate LHRH neuronal
function [36–38]. EGF and TGFα are peptides that signal through the erbB1 receptor. While both can
stimulate LHRH release [9], TGFα has been shown to play a more pivotal role in the regulation of
LHRH neuronal function during puberty in rodents [13,15] and primates [16]. TGFα mRNA and
protein are highly expressed in glial cells and tanycytes in the MBH, their expressions increase
significantly around the time of puberty [39], and pharmacological blockade of the erbB 1 receptor [40]
targeted to the MBH delays puberty [39]. Additionally, sexual maturation induced by hypothalamic
lesions is associated with activation of TGFα expression in glial cells [41,42], and the effect of this
lesion on puberty is blocked by using a selective inhibitor (RG-50864) of TGFα/EGF receptor tyrosine
kinase activity [41]. Also, advanced puberty has been observed in transgenic mice overexpressing the
TGFα gene [43] and in rats carrying grafted cells genetically engineered to secrete TGFα [44]. TGFα
binds to and activates the erbB1/erbB2 receptor complex on adjacent glial cells in MBH. Activation of
these receptors results in the production and release of prostaglandin-E2 (PGE2) from these glial cells,
which then induces prepubertal LHRH secretion upon binding to specific receptors on nearby LHRH
neuron terminals in the median eminence (ME) region of the MBH [22,37]. This is supported by the
fact that the stimulatory effect of TGFα on PGE2 release was blocked by administering an erbB1
receptor antagonist (RG-50864) [37]. Several studies have shown that TGFα stimulates LHRH release
via an indirect mechanism that involves a paracrine effect of this growth factor on glial cells in the
release of LHRH. In this regard, the erbB1 receptors for TGFα have been shown
immunohistochemically only in glial cells [15,16]. Furthermore, Ma et al. [37] have shown in vitro
that the secretion of PGE2 from hypothalamic glial cells is increased after exposure to TGFα and that
the conditioned medium of hypothalamic glial cells treated with TGFα is able to stimulate LHRH
release from GT1 cells, which are immortalized LHRH secreting neurons. Int. J. Environ. Res. Public Health 2011, 8 2878 Int. J. Environ. Res. Public Health 2011, 8 Recently, it has been suggested that some of the hypothalamic effects of ALC are due to
ALC-induced interferences in glial-neuronal signaling networks involved in LHRH secretion. This
review will mainly discuss two emerging glial-neuronal communication networks regarding their
respective relationships to prepubertal LHRH secretion, and then address the actions and interactions
of ALC on these glial-neuronal components during pubertal development. 1. Introduction In this regard, various glial-neuronal adhesion genes have been identified within the hypothalamus
which are not only involved in adhesive interactions, but also mediate intracellular signaling cascades
that are critical for pubertal development [23,24]. The genes of this family include glial receptor
protein tyrosine phosphatase-β (RPTPβ), neuronal contactin and contactin associated protein 1
(Caspr1). Once bound together, this family collectively contributes to glial-neuronal adhesiveness and
to cell to cell communications [25–27]. The interaction between neuronal circuits and glial cells can be
further influenced by metabolic signals, various environmental insults, and drugs of abuse. With
regard to the latter, alcohol (ALC) is a drug of abuse that is known to alter hypothalamic functions that
control reproduction. Chronic ALC exposure has been shown to cause depressed prepubertal LHRH
secretion, and delayed pubertal development in both rodents [28–33] and primates [34,35]. This action
of ALC is important since the onset of mammalian puberty is dependent upon an increase in the
release of LHRH from the basal hypothalamus. 2. EGF/TGFα Family of Growth Factors and Puberty Moreover, in hypothalamic
glial cells, PGE2 formation induced by TGFα and the stimulatory effect of the TGFα treated
conditioned medium on LHRH release are shown to be prevented by the inhibition of erbB receptor
tyrosine kinase activity or prostaglandin synthesis [37,45]. Collectively, these data strongly support the
notion that TGFα acts indirectly in the functional control of neuronal networks governing mammalian
puberty via hypothalamic glial-neuronal communications. Int. J. Environ. Res. Public Health 2011, 8 2879 3. Effects of ALC on the TGFα/erbB1 Receptor/PGE2 Pathway It has been established that ALC acts within the hypothalamus to suppress the release of LHRH in
both prepubertal and adult rats [46,47] and primates [35], and also causes delayed signs of pubertal
maturation in both species [28,34]. Studies to discern the mechanism of this action of ALC to suppress
LHRH release are important for understanding how this drug disrupts pubertal development. An
important component of this ALC effect is PGE2, which plays a major role in the LHRH secretory
process in prepubertal animals [48,49], and is known as a critical factor for glial-dependent regulation
of LHRH release [21,37]. We showed previously [50] that acute ALC alters the EGF/TGFα-erbB1
receptor-COX (cyclooxygenase)-PGE2 pathway by inhibiting the induction of COX, the rate limiting
enzyme necessary for prostaglandin synthesis and lowers prepubertal PGE2 secretion resulting in
suppressed LHRH release [50,51]. Only recently have the mechanisms by which short-term ALC
exposure affects the TGFα-erbB1 receptor -PGE2 pathway been assessed with regard to glial-neuronal
communications within the prepubertal hypothalamus [52]. That study has revealed that short-term
ALC exposure for 4 and 6 days caused an increase in TGFα gene and protein expressions in
prepubertal female rats. The gene expression of TGFα was increased markedly at 4 days (Figure 1). After 6 days of ALC exposure, the level of TGFα gene expression was still modestly but significantly
elevated; however, the levels had declined markedly (not shown) as compared to 4 days of exposure. This effect paralleled an increase in TGFα protein expression at both 4 days (Figure 2A) and 6 days
(Figure 2B). To determine if the elevated hypothalamic levels of TGFα protein were due to an
inhibition of release, we assessed basal TGF〈 secretion from rat MBHs incubated in vitro following 6
days of ALC exposure in vivo. We determined that animals exposed to ALC had suppressed release of
TGFα (Figure 3). Overall, these findings suggest that ALC exposure does not affect transcription or
translation of TGFα, but is capable of interfering with the hypothalamic release of glial TGFα,
resulting in suppressed prepubertal PGE2 and LHRH secretion. Figure 1. Effect of short-term ALC (ethanol) exposure on basal TGFα gene expression in
the MBH of perpubertal female rats. Note that the ALC-treated animals showed an
increase in the gene expression of TGFα compared with control animals. Values represent
mean ± SEM. N = 12–14 animals per group; *** p < 0.001 versus control. 3. Effects of ALC on the TGFα/erbB1 Receptor/PGE2 Pathway 0
1
2
3
4
5
***
Control
ALC (4 day)
Relative expression of
TGF α mRNA 2880 Int. J. Environ. Res. Public Health 2011, 8 Figure 2. Effect of short-term ALC (ethanol) exposure on TGFα protein expression in the
MBH of prepubertal female rats. Composite graphs that show the densitometric
quantitation of the bands corresponding to TGFα protein. These data were normalized to
the internal control β-actin protein, and the densitometric units represent the TGFα/β-actin
ratio. Note that ALC caused an increase in TGFα protein expression on both 4 and 6 days
compared with control animals. Values represent mean ± SEM. N = 7–8 per group. ** p < 0.01; *** p < 0.001 versus control. Figure 2. Effect of short-term ALC (ethanol) exposure on TGFα protein expression in the
MBH of prepubertal female rats. Composite graphs that show the densitometric
quantitation of the bands corresponding to TGFα protein. These data were normalized to
the internal control β-actin protein, and the densitometric units represent the TGFα/β-actin
ratio. Note that ALC caused an increase in TGFα protein expression on both 4 and 6 days
compared with control animals. Values represent mean ± SEM. N = 7–8 per group. ** p < 0.01; *** p < 0.001 versus control. 0.0
0.5
1.0
1.5
2.0
(A)
**
Control
ALC (4 days)
TGFα/β-actin ratio 0.0
0.5
1.0
1.5
2.0
(A)
**
Control
ALC (4 days)
TGFα/β-actin ratio
0.0
0.5
1.0
1.5
2.0
(B)
***
Control
ALC (6 days)
TGFα/β-actin ratio
Figure 3. Effect of short-term in vivo ALC (ethanol) exposure for on TGFα protein
released in vitro from the MBH of prepubertal female rats. Note that TGFα release was
decreased in ALC-treated animals compared with control animals. These data were
normalized to the internal control β-actin protein, and the densitometric units represent
TGFα/β-actin ratio. Values represent mean ± SEM. N = 14 for control, N = 9 for ALC,
** p < 0.01 versus control. (A) (B) Figure 3. Effect of short-term in vivo ALC (ethanol) exposure for on TGFα protein
released in vitro from the MBH of prepubertal female rats. Note that TGFα release was
decreased in ALC-treated animals compared with control animals. These data were
normalized to the internal control β-actin protein, and the densitometric units represent
TGFα/β-actin ratio. Values represent mean ± SEM. N = 14 for control, N = 9 for ALC,
** p < 0.01 versus control. 3. Effects of ALC on the TGFα/erbB1 Receptor/PGE2 Pathway 0.00
0.25
0.50
0.75
1.00
1.25
1.50
**
Control
ALC (6 day)
TGFα released into media
(densitometric units) 0.00
0.25
0.50
0.75
1.00
1.25
1.50
**
Control
ALC (6 day)
TGFα released into media
(densitometric units)
This study also showed that the erbB1 receptor, the principal receptor for TGFα was affected by
ALC. Short-term ALC exposure for 4 and 6 days caused a marked decrease in the synthesis of
the phosphorylated form of the erbB1 receptor at 4 days (Figure 4), with 6 days being almost identical
(not shown), but did not elicit changes in erbB1 gene expression or the synthesis of total,
non-phosphorylated erbB1 protein. It is possible that down regulation of erbB1 gene synthesis had not
yet occurred because of this short-term duration of ALC exposure, but it does appear ALC affected the
phosphorylation of the erbB1 protein. Interestingly, in this study ALC did not affect the synthesis of
total and phosphorylated erbB2, the co-receptor necessary for activation of erbB1 signaling [38] (not This study also showed that the erbB1 receptor, the principal receptor for TGFα was affected by
ALC. Short-term ALC exposure for 4 and 6 days caused a marked decrease in the synthesis of
the phosphorylated form of the erbB1 receptor at 4 days (Figure 4), with 6 days being almost identical
(not shown), but did not elicit changes in erbB1 gene expression or the synthesis of total,
non-phosphorylated erbB1 protein. It is possible that down regulation of erbB1 gene synthesis had not
yet occurred because of this short-term duration of ALC exposure, but it does appear ALC affected the
phosphorylation of the erbB1 protein. Interestingly, in this study ALC did not affect the synthesis of
total and phosphorylated erbB2, the co-receptor necessary for activation of erbB1 signaling [38] (not Int. J. Environ. Res. Public Health 2011, 8 2881 shown), which further demonstrates a specific effect of ALC on the erbB1 receptor. The mechanism of
this action of ALC on erbB1, however, remains unclear. Since TGFα binding initiates the
autophosphorylation of the erbB1 receptor [53,54], it is likely that the ALC-induced impairment of
TGFα release (Figure 3) is a major contributing factor responsible for decreased erbB1
phosphorylation observed in the ALC-treated animals. However, we cannot rule out the possibility
of a direct effect of ALC on the erbB1 autophosphorylation process that is independent of its effect
to suppress TGFα secretion. Int. J. Environ. Res. Public Health 2011, 8 Int. J. Environ. Res. Public Health 2011, 8 Figure 5. Effect of in vivo ALC (ethanol) exposure on prostaglandin-E2 (PGE2) release in
vitro from the MBH of prepubertal rats as determined by enzyme-linked immunoassay. Note that the ALC-treated animals showed a marked decrease in basal PGE2 release
compared with control animals. Values represent mean ± SEM. N = 8 for control, N = 9 for
ALC. ** p < 0.01 versus control. 0
50
100
150
**
Control
ALC (6 day)
pg PGE2 released/mg MBH Studies
in
recent
years
have
identified
upstream
regulatory
sites
in
the
TGFα/erbB1/PGE2/LHRH-secretory pathway. Investigators have shown that the expression of the
POU homeodomain gene, Oct2, increases in the MBH at puberty and that this increase was associated
with the transactivation of TGFα gene expression [59]. Because IGF-1 plays an important role in
control of prepubertal LHRH release, we investigated the possibility that IGF-1 may influence
transcription of the Oct 2 gene and that this gene may be a target of ALC actions [60]. It was
demonstrated that a single injection of IGF-1 to 25 day-old female rats caused increases in both Oct 2a
and c gene transcripts in the MBH and furthermore, showed that an acute dose of ALC (3g/kg) did not
alter basal expression of the gene transcripts, but blocked the IGF-1 induced expressions (Figure 6 a
and b). Similar effects were observed regarding Oct 2c in the POA, but not the Oct 2a transcript. Other
investigators showed an inhibitory effect of chronic ALC exposure on Oct 2 proteins expressed in the
entire rostro-caudal extent of the prepubertal hypothalamus [61]. Interestingly, ALC exposure has also been shown to affect the gene encoding thyroid transcription
factor 1 (TTF1). TTF-1 is a member of the Nkx homeodomain gene family that increases in the
hypothalamus at puberty and has the ability to activate LHRH neurons [38,62]. It was shown that
TTF1 expression peaked at postnatal day 26–27 in controls but that ALC administration beginning at
24 days of age caused suppressed hypothalamic TTF1 protein expression between 25 and 27 days,
which was followed by a significant increase by 30 days [61]; thus, those authors suggested that the
ALC delayed the peak increase in prepubertal TTF1 protein expression. Recently, we observed that
ALC exposure beginning on 27 days of age was associated with an increase in TTF1 gene expression
in the MBH (Figure 7) at 31 days. 3. Effects of ALC on the TGFα/erbB1 Receptor/PGE2 Pathway In this regard, studies have demonstrated that chronic ALC exposure
can disrupt phosphorylation of erbB1 by altering the receptor affinity and/or tyrosine kinase activity
in rats [55,56]. Figure 4. Effect of short-term ALC (ethanol) exposure on phosphorylated erbB1 protein
expressions in the MBH of prepubertal female rats. Note that the ALC-treated animals
showed a marked decrease in phosphorylated erbB1 protein expression compared with
controls. These data were normalized to the total, nonphosphorylated erbB1 protein, and
the densitometric units represent the phosphorylated erbB1/total, non-phosphorylated
erbB1 ratio. Values represent mean ± SEM. N = 8 per group. ** p < 0.01 versus control. 0.00
0.25
0.50
0.75
1.00
1.25
1.50
**
Control
ALC (4 day)
phospho-erbB1/total erbB1 ratio
(densitometric units) 0.00
0.25
0.50
0.75
1.00
1.25
1.50
**
Control
ALC (4 day)
phospho-erbB1/total erbB1 ratio
(densitometric units)
Since it was expected that suppressed erbB1 phosphorylation would result in a downstream
decrease in PGE2 release, animals were chronically exposed to ALC for 6 days and then their
hypothalami removed and incubated in vitro in order to assess the amount of PGE2 released into the
incubation medium. This revealed that ALC exposure suppressed the release of PGE2 (Figure 5), an
action that was associated with the suppressed phosphorylation of the erbB1 receptor shown in
Figure 4. These findings demonstrated for the first time the upstream inhibitory effects of ALC on glial
TGFα/erbB1 pathways that control the production and secretion of PGE2 within the MBH; hence,
providing a mechanism supporting previous reports showing that ALC is capable of suppressing PGE2
and subsequently, LHRH secretion [50,51,57,58]. Since it was expected that suppressed erbB1 phosphorylation would result in a downstream
decrease in PGE2 release, animals were chronically exposed to ALC for 6 days and then their
hypothalami removed and incubated in vitro in order to assess the amount of PGE2 released into the
incubation medium. This revealed that ALC exposure suppressed the release of PGE2 (Figure 5), an
action that was associated with the suppressed phosphorylation of the erbB1 receptor shown in
Figure 4. These findings demonstrated for the first time the upstream inhibitory effects of ALC on glial
TGFα/erbB1 pathways that control the production and secretion of PGE2 within the MBH; hence,
providing a mechanism supporting previous reports showing that ALC is capable of suppressing PGE2
and subsequently, LHRH secretion [50,51,57,58]. 2882 Int. J. Environ. Res. Public Health 2011, 8 This increase at 31 days did not occur in the POA, however, by day
33, both hypothalamic regions showed elevated TTF1 gene expressions compared with controls (not
shown). While additional research is needed in this area, this observation supports the earlier report by
Kim et al. [61], that ALC postponed the prepubertal increase in TTF1 synthesis. 2883 Int. J. Environ. Res. Public Health 2011, 8 Int. J. Environ. Res. Public Health 2011, 8 Figure 6. Effect of acute ALC (ethanol) exposure on IGF-1 induced Oct 2a and 2c in the
MBH of prepubertal female rats. Panels A and B depict densitometric quantitation
corresponding to the Oct 2a and 2c transcripts. IGF-1induced an increase in Oct2a and 2c
mRNA over basal synthesis. Acute ALC exposure did not affect basal synthesis of Oct 2a
and 2c mRNA but blocked the IGF-1 induced synthesis of both Oct 2a and 2c. N = 5–6/group. * p < 0.05. 0.0
0.5
1.0
1.5
2.0
2.5
*
CON
IGF-1
ALC
IGF-1+ALC
*
Oct 2a Oct 2c
A
B
Oct 2 mRNA in MBH
(densitometric units)
Figure 7. Effect of short-term ALC (ethanol) exposure on TTF1 mRNA expression from
the MBH of prepubertal female rats. Note that TTF1 was increased in ALC-treated animals
compared with control animals. Values represent mean ± SEM. N = 9 for control, N = 9 for
ALC, * p < 0.05 versus control. 0.0
0.5
1.0
1.5
2.0
2.5
*
CON
IGF-1
ALC
IGF-1+ALC
*
Oct 2a Oct 2c
A
B
Oct 2 mRNA in MBH
(densitometric units) Figure 7. Effect of short-term ALC (ethanol) exposure on TTF1 mRNA expression from
the MBH of prepubertal female rats. Note that TTF1 was increased in ALC-treated animals
compared with control animals. Values represent mean ± SEM. N = 9 for control, N = 9 for
ALC, * p < 0.05 versus control. 0
1
2
3
*
CON
ALC (4 day)
Relative expression of
TFF-1 mRNA (MBH)
4. Adhesion/Signaling Genes Involved in Glial-Neuronal Communication during Puberty 4. Adhesion/Signaling Genes Involved in Glial-Neuronal Communication during Puberty 4. Adhesion/Signaling Genes Involved in Glial-Neuronal Communication during Puberty There is growing evidence that glial cells can also regulate LHRH secretion by contributing to
plastic arrangements associated with glial-neuronal adhesiveness [13]. Since adhesion/signaling
molecules are abundant in the MBH, and have cell signaling actions [23,63], it has been suggested that
they play a role in intracellular communication between glial cells and LHRH neuron terminals [64,65]. In this regard, several genes have been identified which synthesize adhesion/signaling proteins
responsible for the structural integrity of bi-directional glial-neuronal communication. The synaptic
cell adhesion molecule (SynCAM1) expressed by both glia and neuronal cells, promotes the
glial-neuronal adhesiveness via homophylic, extracellular domain-mediated interactions required for
synaptic assembly [13]. Similarly, another report using an in vitro adhesion assay shows that both
hypothalamic glial cells and GT1-7 neuronal cells adhere to the extracellular domain of SynCAM1 and
suggest the importance of this adhesion molecule in cell-cell communication between glial cells and
LHRH neurons [64,65]. SynCAM1 is also associated with neuregulin activated erbB4 receptor, one of Int. J. Environ. Res. Public Health 2011, 8 2884 the glial cell erbB receptors involved in LHRH secretion at puberty [64,66] and is expressed in
hypothalamic glial cells. Specifically, erbB4 receptor forms a heterodimer complex with erbB2 and
activation by neuregulins causes LHRH release via an action involving glial PGE2 release and
facilitation of erbB-1 mediated signaling events [66,67]. Disruption of erbB4/-2 signaling results in
delayed puberty and an inhibition of SynCam1 expression in hypothalamic glial cells [66,67]. Futhermore, a study has shown that transgenic mice with a double negative form of SynCam1 lacking
the intracellular domain causes these animals to have delayed puberty [68]. These observations suggest
that one of the mechanisms underlying erbB4 receptor facilitation of LHRH neuronal function
involves SynCAM1 dependent signaling during pubertal development. The effect of ALC exposure on
SynCAM1 gene and protein expression is under investigation. Another example among the adhesion/signaling genes within the MBH is a three member family
consisting of neuronal contactin associated protein-1 (Caspr1), a transmembrane protein that binds to
contactin on the same neuronal cell membrane. The contactin portion of this Caspr1/contactin complex
is bound by a glial transmembrane protein, receptor protein tyrosine phosphatase beta (RPTPβ); thus,
forming the three member assembly that can contribute to glial-neuronal adhesiveness [25,26]. Contactin participates in axonal growth, synaptogenesis and neuroendocrine function, and contactin
expression in hypothalamic secretory neurons has been shown to be altered in response to changes in
glial-neuronal associations [27]. 4. Adhesion/Signaling Genes Involved in Glial-Neuronal Communication during Puberty Caspr1 is proposed as a signaling molecule of contactin, which
mediates its cell adhesion by binding to RPTPβ and activating intracellular signaling pathways in
neurons [63]; thus, following binding, this three member assembly contributes to bidirectional cell-cell
communications between glial and neuronal cells associated with hypothalamic neuroendocrine
functions [27]. It is important to note that LHRH neurons express both contactin and Caspr1 [23]. It
has been suggested that upon binding together this system not only provides adhesiveness between
glial connections with LHRH terminals, but also regulates intracellular processes [64]. Interestingly,
the intimate association between glial “end feet” and LHRH neuron terminals with the ME area
is modified by the actions of different reproductive factors [69–71], in that changes in the contact
between glia and neurons fluctuates depending on steroid milieu and stage of pubertal
devleopment [72]. Taken together, a concept is emerging that suggests that these glial-neuronal
molecules facilitate hypothalamic neurosecretion via changes in the arrangement of glial-neuronal
adhesion and signaling that could alter LHRH release and the pubertal process. 5. Effects of ALC on Glial-Neuronal Adhesion and Signaling In addition to ALC affecting neuronal inputs involved in prepubertal LHRH secretion, there has
been some recent attention given to the actions of this drug on the contactin-Caspr1-RPTPß signaling
system as it relates to glial-LHRH neuronal interactions [73]. In this regard, short-term ALC exposure
caused a marked decrease in the basal expression of the RPTPβ gene (Figure 8A), but did not affect
the expression of either contactin (Figure 8B) or Caspr1 (Figure 8C). Similarly, ALC caused
suppressed levels of the RPTPβ protein (Figure 9A), with the expressions of both contactin (Figure 9B)
and Caspr1 (Figure 9C) proteins being unaltered. The ALC-induced decrease in glial RPTPβ gene
expression in this study indicates a reduced amount of peptide available for binding to the
contactin-Caspr1 complex on LHRH neurons. As a result, decreased adhesiveness between the glia Int. J. Environ. Res. Public Health 2011, 8 2885 and LHRH neurons in the MBH following ALC exposure would be expected, an action that could
affect neuronal functions associated with pubertal development. The binding of glial RPTPβ to the
contactin/Caspr1 complex on neuron terminals initiates the cell adhesion and subsequently, activates
neuronal intracellular signaling pathways [63]. The precise contribution of contactin-Caspr1 signaling
to LHRH secretion remains to be determined, but previous studies have suggested that a function of
the RPTPβ-contactin-Caspr1 family is to facilitate neurosecretion through changes in glial-neuronal
signaling [23,27]. The cell adhesiveness component also promotes a more secure proximity for
glial-derived secretions to bind to their receptors on the LHRH nerve terminals. Whatever the
mechanism, the fact that ALC alters the synthesis of prepubertal glial RPTPβ in the hypothalamus,
which is required for binding to the neuronal contactin/Caspr1 complex, suggests diminished
glial-neuronal adhesiveness and thus, altered facilitation of LHRH release by the products secreted
from the neighboring glial cells. Figure 8. Effect of short-term ALC (ethanol) exposure on the gene expressions of basal
RPTPβ (A), Contactin (B) and Caspr1(C) in the MBH of prepubertal female rats. Note that
ALC caused a marked decrease in the gene expression of basal RPTPβ compared with
control animals. Values represent mean ± SEM. N = 12–13 per group. *** p < 0.001
versus control. 0.0
0.5
1.0
1.5
2.0
2.5
***
RPTP? Contactin Caspr1
(A)
(B)
(C)
CON
ALC
Relative expression of mRNA
(densitometric units)
Figure 9. ns and Interactions of IGF-1 and ALC on Glial-Neuronal Adhesion and Signaling: IGF-1 plays a critical role in the pubertal process. Initially, it was shown that the peptide is capable
of inducing release of LHRH from the prepubertal hypothalamus in vitro [10], then later demonstrated
thast IGF-1 acts within the hypothalamus to accelerate the timing of female puberty [11]. Subsequently,
it was show that premature elevation of serum IGF-1 advanced first ovulation in rhesus monkeys [74]. While IGF-1 is produced by neurons and glia in the MBH, the majority of IGF-1 present in this region
during puberty is derived from peripheral sources, such as liver [11,75,76]. IGF-1 binds to type 1 IGF
receptors (IGF-1R) in different tissues including the brain, where the greatest concentration of IGF-1R
has been observed in the ME of both rats [77] and primates [78]. Recently, DiVall et al. [79] showed
that transgenic mice lacking the IGF-1R on their LHRH neurons have delayed puberty, further
supporting the importance of this peptide for pubertal development. IGF-1 is also capable of acting at
both glial and neuronal levels involved in neuroendocrine events within the hypothalamus in
association with prepubertal LHRH secretion [47]. These findings suggest that IGF-1 may play a role
in regulating the expression of adhesion and signaling molecules. Interestingly, we showed that IGF-1
induced an increase in the expression of the RPTPß gene in prepubertal female rat hypothalamus
(Figure 10); however, it did not affect the gene expression of either contactin or Caspr1 [73]. Glial
RPTPß is expressed abundantly within the MBH [80]. The knowledge that increased circulating levels
of IGF-1 at puberty can cross the blood brain barrier and enter the MBH region [11], and that the
circulating peptide is taken up by hypothalamic glia in the MBH [81], suggests that the
IGF-1-induced expression of RPTPß in the MBH may facilitate neurosecretion via changes in
glial-neuronal adhesive signaling. The fact that IGF-1 is important for LHRH release at puberty [10,11] and the observation that ALC
can alter prepubertal IGF-1 signaling [29] suggests that ALC could have a detrimental impact on
pubertal development by interfering with glial neuronal signaling networks. In support of this, it has
been shown recently [73] that ALC blocked the ability of IGF-1to induce prepubertal RPTPβ gene
expression (Figure 10). 6. Actions and Interactions of IGF-1 and ALC on Glial-Neuronal Adhesion an 6. Actions and Interactions of IGF-1 and ALC on Glial-Neuronal Adhesion and Sig 5. Effects of ALC on Glial-Neuronal Adhesion and Signaling Effect of short-term ALC (ethanol) exposure on the protein expressions of basal
RPTPβ (A), contactin (B) and Caspr1(C) in the MBH of prepubertal rats. Note that the
protein expression of RPTPβ was markedly decreased in ALC exposed animal; however,
the protein expressions of contactin and Caspr1 were unaltered. These data were
normalized to the internal control β-actin protein, and the densitometric units represent the
respective specific protein/β -actin ratio. Values represent mean ± SEM. N = 10 for control,
N = 6 for ALC. *** p < 0.001 versus control. e 9. Effect of short-term ALC (ethanol) exposure on the protein expressions of basal Figure 9. Effect of short-term ALC (ethanol) exposure on the protein expressions of basal
RPTPβ (A), contactin (B) and Caspr1(C) in the MBH of prepubertal rats. Note that the
protein expression of RPTPβ was markedly decreased in ALC exposed animal; however,
the protein expressions of contactin and Caspr1 were unaltered. These data were
normalized to the internal control β-actin protein, and the densitometric units represent the
respective specific protein/β -actin ratio. Values represent mean ± SEM. N = 10 for control,
N = 6 for ALC. *** p < 0.001 versus control. Figure 9. Effect of short-term ALC (ethanol) exposure on the protein expressions of basal
RPTPβ (A), contactin (B) and Caspr1(C) in the MBH of prepubertal rats. Note that the
protein expression of RPTPβ was markedly decreased in ALC exposed animal; however,
the protein expressions of contactin and Caspr1 were unaltered. These data were
normalized to the internal control β-actin protein, and the densitometric units represent the
respective specific protein/β -actin ratio. Values represent mean ± SEM. N = 10 for control,
N = 6 for ALC. *** p < 0.001 versus control. 0.00
0.25
0.50
0.75
1.00
Contactin/β-actin ratio
(densitometric units)
0.00
0.25
0.50
0.75
1.00
***
CON
ALC (6 day)
RPTPβ/β-actin ratio
(densitometric units)
0.00
0.25
0.50
0.75
1.00
Caspr1/β-actin ratio
(densitometric units)
(A)
(B)
(C) 0.00
0.25
0.50
0.75
1.00
Contactin/β-actin ratio
(densitometric units)
0.00
0.25
0.50
0.75
1.00
***
CON
ALC (6 day)
RPTPβ/β-actin ratio
(densitometric units)
0.00
0.25
0.50
0.75
1.00
Caspr1/β-actin ratio
(densitometric units)
(A)
(B)
(C) C
t
ti /β
ti
ti
0.00
0.25
0.50
0.75
1.00
***
CON
ALC (6 day)
RPTPβ/β-actin ratio
(densitometric units)
(A) Int. J. Environ. Res. Public Health 2011, 8 2886 Int. J. Environ. Res. Public Health 2011, 8 Int. J. Environ. Res. Public Health 2011, 8 Figure 10. Effect of acute ALC exposure on basal and IGF-1 stimulated gene expressions
of RPTPß in the MBH of prepubertal female rats. Note that IGF-1 increased the gene
expression of RPTPβ compared with the basal level of control. Importantly, the basal
expression of the RPTPβ gene was not altered by ALC alone, but the IGF-1 induced
expression of RPTPβ was blocked by the presence of ALC. Values represent mean ± SEM. N = 8–10 per group. ** p < 0.01 versus control and ALC + IGF-1. 0.0
0.5
1.0
1.5
2.0
2.5
**
CON
IGF-1
ALC
ALC+IGF-1
Relative expression of
RPTPβ mRNA
Conclusions ns and Interactions of IGF-1 and ALC on Glial-Neuronal Adhesion and Signaling: The mechanism by which ALC influenced the IGF-1 induced expression of
RPTPβ remains to be determined; however, this effect could be due to an action of ALC at the level of
the IGF-1R and/or to an altered post-receptor event. Interestingly, ALC administration does not alter
IGF-1R gene or protein expression [29], although we cannot rule out that it may affect mechanisms
regulating receptor function. This suggests that ALC may act on a pathway component downstream
from the IGF-1R. In this regard, chronic ALC administration was shown to suppress the basal protein
expression of phosphorylated Akt, a transduction signal activated by IGF-1 [82]. Acutely, ALC was
shown to block the IGF-1 induced expression of other genes involved in the pubertal process [60,83],
and that this occurs by blocking the peptides ability to induce phosphorylation of Akt [83]. Chronic
exposure to ALC has been shown to decrease circulating levels of IGF-1 in prepubertal rats [29] and
rhesus monkeys [34]; actions associated with altered pubertal development. Based on these collective
results, it is suggested that the decreased prepubertal levels of circulating IGF-1 available to the MBH,
as well as altered post-receptor transduction signals, may contribute to the ability of ALC to cause the
suppression in RPTPβ gene expression. 2887 Int. J. Environ. Res. Public Health 2011, 8 2888 Int. J. Environ. Res. Public Health 2011, 8 Figure 11. Schematic drawing showing glial-LHRH neuronal associations and sites of
ALC effects on RPTPβ in the median eminence of juvenile female rats. For clarity, details
of other downstream pathways in this region are not shown. ALC, alcohol; BBB, blood
brain barrier; Caspr1, contactin associated protein-1; EGF, epidermal growth factor; erbB1,
EGF/TGF receptor; ER, estrogen receptor; IGF-1, insulin-like growth factor-1; IGFR,
Insulin-like growth factor-1 receptor; LHRH, luteinizing hormone releasing hormone;
PGE2, prostaglandin-E2; PGER, prostaglandin-E2 receptor; RPTPβ, receptor protein
tyrosine phosphatase β; TGF, transforming growth factor [73]. Figure 11. Schematic drawing showing glial-LHRH neuronal associations and sites of
ALC effects on RPTPβ in the median eminence of juvenile female rats. For clarity, details
of other downstream pathways in this region are not shown. ALC, alcohol; BBB, blood
brain barrier; Caspr1, contactin associated protein-1; EGF, epidermal growth factor; erbB1,
EGF/TGF receptor; ER, estrogen receptor; IGF-1, insulin-like growth factor-1; IGFR,
Insulin-like growth factor-1 receptor; LHRH, luteinizing hormone releasing hormone;
PGE2, prostaglandin-E2; PGER, prostaglandin-E2 receptor; RPTPβ, receptor protein
tyrosine phosphatase β; TGF, transforming growth factor [73]. cknowledgements
This work was supported by the NIH grant AA07216 (to WLD). The authors declare no conflict
interest. Acknowledgements This work was supported by the NIH grant AA07216 (to WLD). The authors declare no conflict
of interest. This work was supported by the NIH grant AA07216 (to WLD). The authors declare no conflict
of interest. 7. Conclusions It is becoming increasingly clear that glial-neuronal interactions play an important role in
prepubertal LHRH secretion and the subsequent acquisition of female pubertal development. In this
review, we have mainly discussed the actions of glial-neuronal communication networks at puberty
and how these actions are influenced by ALC, a drug of abuse known to alter pubertal development. A summary of these cellular communications and the sites of ALC actions are shown in Figure 11. One of these communication systems is the TGFα-erbB1 receptor signaling system. We have made
reference to research indicating that glial TGFα activates the erbB1/erbB2 receptor complex on
adjacent glia in the MBH. This activation causes a cascade of events leading to the increased synthesis
and release of PGE2, which then binds to its receptor on nearby LHRH neuron terminals causing
stimulated release of the peptide. Additionally, evidence was presented showing that ALC is capable
of interfering with hypothalamic glial to glial signaling involved with prepubetal PGE2 sythesis/release. The other communication network discussed in detail is the contactin-Caspr1 complex that binds to
glial RPTPβ. After binding together, the newly formed three member family provides adhesiveness
and promotes signaling between glia and LHRH nerve terminals in the MBH. Interestingly, we
provided evidence that ALC exposure can interfere with this glial-neuronal communication family at
puberty by suppressing the synthesis of glial RPTPβ. Additionally, we discussed how IGF-1 can
influence both PGE2 and RPTPβ signaling. Overall, this review further indicates that glial-neuronal
communications are important for LHRH secretion at puberty, and that ALC is capable of altering
these cell-cell interactions. There are obviously other glial-neuronal systems not discussed here
that deserve further investigation into their respective roles in neuroendocrine secretion at puberty,
as well as determining whether they are affected by endocrine disruptive substances that may alter
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LJMU Research Online Nie, J, George, KP, Duan, F, Tong, TK and Tian, Y Nie, J, George, KP, Duan, F, Tong, TK and Tian, Y Histological evidence for reversible cardiomyocyte changes and serum
cardiac troponin T elevation after exercise in rats. LJMU Research Online Article Citation (please note it is advisable to refer to the publisher’s version if you
intend to cite from this work) Nie, J, George, KP, Duan, F, Tong, TK and Tian, Y (2016) Histological
evidence for reversible cardiomyocyte changes and serum cardiac troponin
T elevation after exercise in rats. Physiological Reports, 4 (24). ISSN 2051-
817X LJMU has developed LJMU Research Online for users to access the research output of the
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access may require a subscription. For more information please contact researchonline@ljmu.ac.uk http://researchonline.ljmu.ac.uk/ ORIGINAL RESEARCH
Histological evidence for reversible cardiomyocyte changes
and serum cardiac troponin T elevation after exercise in
rats
Jinlei Nie1, Keith George2, Fei Duan3, Tomas K. Tong4 & Ye Tian5
1 School of Physical Education and Sports, Macao Polytechnic Institute, Macao, China
2 Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, United Kingdom
3 College of Basic Medical Sciences, Hebei University, Hebei, China
4 Dr. Stephen Hui Research Centre for Physical Recreation and Wellness, Department of Physical Education, Hong Kong Baptist University,
Hong Kong, China
5 China Institute of Sport Science, Beijing, China
Physiological Reports ISSN 2051-817X doi: 10.14814/phy2.13083 Physiol Rep, 4 (24), 2016, e13083,
doi: 10.14814/phy2.13083 running, can result in cTn appearance during exercise or
recovery (Middleton et al. 2008; Shave et al. 2010; Gres-
slien and Agewall 2016). Postexercise cTn levels have risen
above the clinical threshold values used to identify AMI
in a number of studies (Shave et al. 2010; Gresslien and
Agewall 2016) presenting something or a diagnostic
dilemma. Histological evidence for reversible cardiomyocyte changes
and serum cardiac troponin T elevation after exercise in Jinlei Nie1, Keith George2, Fei Duan3, Tomas K. Tong4 & Ye Tian5 1 School of Physical Education and Sports, Macao Polytechnic Institute, Macao, China 2 Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, United Kingdom
3 College of Basic Medical Sciences, Hebei University, Hebei, China
4 Dr. Stephen Hui Research Centre for Physical Recreation and Wellness, Department of Physical Education, Hong Kong Baptist University,
Hong Kong, China 5 China Institute of Sport Science, Beijing, Chin Physiological Reports ISSN 2051-817X ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society.
This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
2016 | Vo ORIGINAL RESEARCH Histological evidence for reversible cardiomyocyte changes
and serum cardiac troponin T elevation after exercise in
rats Correspondence Correspondence
Jinlei Nie, School of Physical Education and
Sports, Macao Polytechnic Institute, Rua de
Luis Gonzaga Gomes, Macao, China. Tel: +853-8559 6832
Fax: +853-2851 8538
E-mail: jnie@ipm.edu.mo Correspondence
Jinlei Nie, School of Physical Education and
Sports, Macao Polytechnic Institute, Rua de
Luis Gonzaga Gomes, Macao, China. Tel: +853-8559 6832
Fax: +853-2851 8538
E-mail: jnie@ipm.edu.mo The Physiological Society and the American Physiological Society. Keywords Cardiac biomarker, exercise, myocardium. This study characterized cardiac troponin T (cTnT) appearance and associated
histological evidence of reversible or irreversible changes in myocardial ultra-
structure, determined via electron microscopy, in rats undertaking isopro-
terenol (ISO) infusion or an endurance exercise challenge. Male rats were
randomized into ISO and exercise groups. In ISO trials rats were killed 5 h
(ISO-5H) and 24 h (ISO-REC19H) after a single ISO or saline injection (SAL-
5H; SAL-REC19H). In the exercise trials rats were killed before, as a control
(EXE-CON), immediately after (EXE-END5H) and 19 h after (EXE-REC19H)
a 5-h bout of swimming with 5% body weight attached to their tail. Serum
cTnT was quantified by electrochemiluminescence, and myocardial samples in
ISO-REC19H, EXE-REC19H and SAL-REC19H were harvested for assessment
of specific mitochondrial injury scores using electron-microscopy. cTnT was
undetectable in all control animals (SAL-5H/SAL-REC19H and EXE-CON). cTnT increased in all animals after ISO and exercise but the response was sig-
nificantly higher (P < 0.05) at ISO-5H (median [range]: 2.60 [1.76–6.18]
lg L1) than at EXE-END5H (median [range]: 0.05 [0.02–0.14] lg L1). cTnT returned to baseline at EXE-REC19H, but had not completely recovered
at ISO-REC19H (median [range]: 0.17 [0.09–1.22] lg L1). Mitochondrial
“injury scores” were significantly higher (P < 0.05) in ISO-REC19H compared
to EXE-REC19H and SAL-REC19H, with no difference between EXE-REC19H
and SAL-REC19H. Mitochondria from EXE-REC19H appeared aggregated in
nonlinear clusters in a small number of scans. These findings suggest that
acute exercise-induced appearance of cTnT in this animal model is only asso-
ciated with reversible changes in cardiomyocyte structure. This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Funding Information Funding Information
The study was supported by a research grant
from Macao Polytechnic Institute (RP/ESEFD-
01/2012). Received: 12 November 2016; Revised: 22
November 2016; Accepted: 23 November
2016 Received: 12 November 2016; Revised: 22
November 2016; Accepted: 23 November
2016 doi: 10.14814/phy2.13083 Experimental protocol The rat ISO-treated protocol and exercise model were
performed according to Goldspink et al. (2004) and Chen
et al. (2000), respectively. All sessions for ISO and exer-
cise groups started at 1000 h. On an experimental day,
food was removed at 0800 h and returned to provide the
rat chow and water ad libitum from 1500 h. The ISO
groups consisted of 36 rats, which were treated with a
single dose of either normal saline (10 mL kg1 body
weight, controls; n = 16), or ISO (5 mg kg1 body
weight; n = 20) injected subcutaneously into rats. The
rats in the ISO trial were killed after 5 h (ISO-5H, n = 8)
and 24 h (ISO-REC19H, n = 8) after injection. Four rats
were removed from this protocol due to early death. The
control rats were killed 5 h (SAL-5H, n = 8) and 24 h
(SAL-REC19H, n = 8) after injection. The exercise groups
consisted of 30 rats: eight rats were used as nonexercise
controls (EXE-CON) and killed at rest, 22 rats swam for
5 h in a swimming tank (temperature 35°C, depth
30 cm) with 5% body weight attached to their tail. Rat
swimming with a 5% load is considered a high intensity
exercise (>80% VO2max) (McArdle and Montoye 1966;
McArdle 1967). A 180 liters water tank was divided into
four lanes with a surface area of 30 9 30 cm and a water
depth of 35 cm per lane to allow individual swim exer-
cise, and the water maintained at 35°C. The rats main-
tained
continuous
swimming
involves
continuous
movement of the rats’ forelimbs and hindlimbs while
maintaining its snouts above the waterline. Six rats failed
to complete the swim protocol. After swimming, the rats
were towel dried and killed immediately (EXE-END5H,
n = 8), and 19 h after swimming (EXE-REC19H, n = 8). Consequently, the purpose of this study was to observe
cTnT appearance and histological evidence of reversible
or irreversible damage to the structure of cardiomycoytes
in a rat model of prolonged exercise in comparison to a
standard ISO infusion protocol. Data were also compared
to control data (prior to exercise and with saline infu-
sion) and data were assessed immediately and after 19 h
of recovery postexercise. Introduction Cardiac troponins (cTn: cTnT and cTnI) are highly speci-
fic and sensitive markers of myocardial cell damage and
have been adopted as the gold standard for the biochemi-
cal
detection
of
cardiomyocyte
insult
during
acute
myocardial
infarction
(AMI)
(Collinson
et al. 2003). Somewhat
counterintuitively
numerous
studies
have
demonstrated that prolonged exercise, such as marathon The clinical interpretation of postexercise cTn release is
complex, but should be interpreted holistically with other 2016 | Vol. 4 | Iss. 24 | e13083
Page 1 J. Nie et al. Heart Cell Changes and Blood cTnT after Exercise J. Nie et al. clinical signs and symptoms of cardiovascular disease. Despite this, case reports of sudden cardiac death in ath-
letes with adverse cardiac structural remodeling as well as
reports of links between cTn elevation and depressed car-
diac function (La Gerche 2013; Eijsvogels et al. 2016)
have prompted ongoing debate about the cardiovascular
consequences of high levels of exercise exposure (Whyte
et al. 2009). Whether exercise-induced cTn release reflects
reversible or irreversible changes in cardiomyocyte integ-
rity is not clear and is currently the source of some
debate (George et al. 2011; Guasch and Nattel 2013;
Eijsvogels et al. 2016). ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. Animals Sixty-six male Sprague–Dawley (SD) rats (12 weeks old),
weighing 210–230 g, were used in these experiments. The
rats were housed in cages in rooms regulated for tempera-
ture (21–23°C), humidity (40–55%), and light cycle
(0700–1700) and provided laboratory rat chow and water
ad libitum. The research involving rodents in this study
conforms to the National Institutes of Health Guidelines
for the Care and Use of Laboratory Animals and was
approved by the Institutional Animal Care and Use
Committee. Evidence to determine whether exercise-induced cTn
release is associated with reversible or irreversible car-
diomyocyte damage is not available in humans. Histo-
logical changes in cardiomyocyte structure, associated
with elevated serum cTn, can be determined in animal
models (Chen et al. 2000) in association with the appli-
cation of controlled exercise doses. To our knowledge,
Chen et al. (2000) and Olah et al. (2015) are the only
two
groups
to
synchronously
evaluate
histological
changes
in
cardiomyocyte
structure
and
postexercise
serum cTn levels in animal experiments. These studies
reported an elevation in cTnT as well as hematoxylin–
eosin staining evidence of sporadic cardiomyocyte dam-
age after prolonged exercise. Despite this the authors
admitted that the subtle nature of myocardial injury
may not always be identifiable by limited light micro-
scopic examination (Chen et al. 2000; Olah et al. 2015). The authors also concluded that more detailed studies
are required as there is a clear diagnostic and prognostic
distinction between irreversible and reversible myocardial
injury to cardiomyocytes. Detailed electron microscopy
has been commonly used to identify myocardial injury
in animal experiments (Ishikawa et al. 1997) and isopre-
naline (ISO) infusion is a common experimental model
of irreversible cardiomyocyte injury (Goldspink et al. 2004). Similarly
this
model
has
been
employed
to
explore the relationship between cTn release and histo-
logical changes in cardiomyocyte structure in rats (Acikel
et al. 2003). Statistical analyses For analysis of serum cTnT, a third-generation assay was
performed using an electrochemiluminescence technology
employed by the Elecsys 2010 automated batch analyzer
(Roche Diagnostics, Basel, Switzerland). This assay has
been previously validated for use in several laboratory
animals, including rats (Fredericks et al. 2001). The
interassay
coefficient
of
variation
was
10%
at
0.03 lg L1 and 3% at 0.10 lg L1. The intraassay
coefficient of variation was 8% at 0.03 lg L1 and 2%
at 0.10 lg L1, with a detection limit of 0.01 lg L1
(which coincides with the 99th percentile value) and an
upper limit of 25 lg L1. Serum cTnT reported as
<0.01 lg L1 was represented as 0.005 lg L1 (Cleave
et al. 2001; Nie et al. 2011b). All cTnT analyses were per-
formed with the same assay kit. Before the assays were
performed, the analyzers were calibrated with standard
calibrators according to the manufacturer-recommended
protocols. The Kolmogorov–Smirnov test was used to evaluate the
normality of the data distribution. Due to the skewed dis-
tribution of cTnT data were expressed as median and
range and we applied a nonparametric Kruskal–Wallis H
test to compare the serum cTnT across time points. A
Mann–Whitney U-test was applied post hoc to assess
pairwise comparisons where appropriate. The rates of Table 1. Severity and extent scoring of mitochondria injury. Scores
Features
Severity scoring1
0
Intact double membrane, compact orderly christae, and a
homogeneous dense matrix
1
Mitochondrial swelling
2
Lysis and breakage of mitochondrial cristae (i.e., cristolyis)
3
Mitochondrial matrix proteins disintegrate
4
Large dense granules in the mitochondrial matrix
5
Ruptured and fragmented mitochondria
Extent scoring
0
Fully intact
1
Involvement of single scattered myofibers
2
Involvement of groups of myofibers
3
Focal groups
4
Confluent groups
1The pathological descriptors of each ascending severity score are
additional features to those of the lower scores. Table 1. Severity and extent scoring of mitochondria injury. Scores
Features
Severity scoring1 Table 1. Severity and extent scoring of mitochondria injury. Tissue preparation Rats were anesthetized with sodium pentobarbital (Nem-
butal,
50 mg kg1
ip). The
abdominal
cavity
was
quickly opened and ~5 mL of blood was drawn from the
abdominal aorta. Serum samples were collected and
stored at 80°C for cTnT measurements. The chest cavity
was then quickly opened and the heart was excised. Left
ventricular tissues were removed and immediately placed
in glutaraldehyde. Experimental protocol The study of cTnT but not cTnI
ensures comparability between our results and those of
other related studies, given only a single cTnT assay from
diagnostic manufacturer (Rowland et al. 2010) is com-
mercially available (Shave et al. 2007). It has been shown that peak serum cTnT occurs at
4–6 h after ISO injection in rats (O’Brien et al. 2006). Similarly, serum cTnT concentrations in animals (Chen
et al. 2000) and humans (Nie et al. 2011a; Tian et al. 2012) peaked ~5–6 h after the initiation of exercise, 2016 | Vol. 4 | Iss. 24 | e13083
Page 2 Heart Cell Changes and Blood cTnT after Exercise J. Nie et al. independent of duration. Consequently we believe we
employed a suitable design to obtain coincident peak
comparisons of serum cTnT by drawing blood samples
5 h after ISO injection or the initiation of exercise. In
addition, given that the peak histological severity (myode-
generation and necrosis) does not typically occur until
18–24 h after exercise (Chen et al. 2000) or ISO injection
(Ishikawa et al. 1997; Goldspink et al. 2004), hearts from
EXE-REC19H (n = 8) and ISO-REC19H (n = 8), along
with controls (SAL-REC19H, n = 8), were selected for
histological examination. a diamond knife. Preliminary sections were stained with
alkaline toluidine blue, and appropriate areas were chosen
for sectioning. Random sections were collected on 200-
mesh copper grids, stained with uranyl acetate and lead
citrate, coated with carbon, and examined in an electron
microscope (JEM-1230, JEOL Ltd, Tokyo, Japan). p
y
p
According to the previous work (Ishikawa et al. 1997;
Cheville 2009), mitochondrial quality is the key to distin-
guishing reversible and irreversible cell injury. Specifically,
intact
mitochondria
are
characteristic
of
reversible
changes, whereas irreversible changes included disintegra-
tion of mitochondrial cristae and matrix proteins and
accumulation of large dense granules accumulate in the
matrix. These changes can lead to cell death if critical
numbers of mitochondria are affected (Cheville 2009). A
mitochondrial “injury scoring” system was adapted from
Rahman et al. (1982) and Cheville (2009). Evaluation was
made of 3+ blocks per heart and scoring was tabulated
individually for each of 20 grid squares of tissue section
using the criteria presented in Table 1. A severity and
extent score was produced for each grid square for each
rat. The pathologist scoring the sections was blind to the
groups. ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. Histological examination Myocardial samples for electron microscopic examination
were minced into blocks (~1 mm per side), fixed in glu-
taraldehyde
(30 mL L1
in
0.1 mol L1
sodium
cacodylate buffer, pH 7.4), postfixed in 10 g L1 osmium
tetroxide, dehydrated in ethanol, embedded in Spurr’s
low-viscosity resin, and sectioned with a Sorvall Porter-
Blum ultramicrotome (Leica Co., Wetzlar, Germany) and 2016 | Vol. 4 | Iss. 24 | e13083
Page 3 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. Heart Cell Changes and Blood cTnT after Exercise J. Nie et al. mitochondrial remnants (score 5) (Fig. 3). The mitochon-
drial appearance of EXE-REC19H was similar to that of
SAL-REC19H with only a small number of scans demon-
strating mitochondrial aggregation in clusters (Fig. 4) and
occasional mitochondrial swelling (score of 1). cTnT-positive (the percentage of subjects with cTnT
exceeding the detection limit of 0.01 lg L1) at each
assessment point were compared using Fisher’s exact test. One-way analysis of variance (ANOVA) was employed
to examine the differences in histological mitochondrial
“injury scoring” among ISO, exercise and control groups. Post hoc analyses using Newman–Keuls were performed
for cases in which the main effect was significant. Statisti-
cal significance was assumed at a level of P < 0.05. Data
analysis was performed using the statistical software pack-
age SPSS 11.5 (SPSS Inc., Chicago, IL). Figure 1. Individual data points for cardiac troponin T (cTnT) in
isoprenaline-treated (A) and exercise (B) rats. ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. Results Data for serum cTnT in ISO/Saline trials and exercise are
presented in Table 2 and as individual data points in Fig-
ure 1. cTnT was undetectable in all animals at SAL-5H/
SAL-REC19H and EXE-CON. Serum cTnT was signifi-
cantly (P < 0.05) increased in all individuals, and as a
cohort compared to respective controls, at ISO-5H and
EXE-END5H, but the response was significantly higher
(P < 0.05) at ISO-5H. Thereafter, cTnT returned to base-
line at EXE-REC19H (vs. EXE-CON P > 0.05), but not
completely at ISO-REC19H. Correspondingly, the positive
rate of cTnT was significantly higher at ISO-REC19H
than EXE-REC19H (P < 0.05). Histological evidence in SAL-REC19H showed mito-
chondria were intact (Fig. 2). Cardiac tissue from ISO
treated rats (ISO-REC19H) revealed disrupted mitochon-
dria in both severity and extent when compared with the
exercise
(EXE-REC19H)
and
control
(SAL-REC19H)
groups (Table 3). The mitochondrial injury scores were
not
different
between
the
EXE-REC19H
and
SAL-
REC19H. The mitochondria from ISO-REC19H samples
demonstrated cristae and matrix proteins disintegration
(scores 2–3), large dense granules accumulate in the
matrix
(score
4),
and
ruptured
and
fragmented Figure 1. Individual data points for cardiac troponin T (cTnT) in
isoprenaline-treated (A) and exercise (B) rats. Figure 1. Individual data points for cardiac troponin T (cTnT) in
isoprenaline-treated (A) and exercise (B) rats. Table 2. Serum cardiac troponin T in ISO-treated and EXE rats 5 h (5H) and 24 h (recovery) after the onset of intervention (ISO injecting or
exercise), and corresponding CON. CON
5H
Recovery
ISO
Median (range, lg L1)
0.005 (–)
2.60 (1.76–6.18)1 2
0.17 (0.09–1.22)1 2
Positive rate3
0/8
8/81
8/81 2
EXE
Median (range, lg L1)
0.005 (–)
0.05 (0.02–0.14)1
0.005 (0.005–0.02)
Positive rate3
0/8
8/81
1/8
ISO, isoprenaline; EXE, exercise; CON, control. 1Significantly different from corresponding CON value, P < 0.05. 2Significantly different from corresponding EXE value, P < 0.05. 3Values presented as number of positive/total observations. Table 2. Serum cardiac troponin T in ISO-treated and EXE rats 5 h (5H) and 24 h (recovery) after the onset of intervention (ISO injecting or
exercise), and corresponding CON. CON
5H
Recovery
ISO
Median (range, lg L1)
0.005 (–)
2.60 (1.76–6.18)1 2
0.17 (0.09–1.22)1 2
Positive rate3
0/8
8/81
8/81 2
EXE
Median (range, lg L1)
0.005 (–)
0.05 (0.02–0.14)1
0.005 (0.005–0.02)
Positive rate3
0/8
8/81
1/8 Table 2. Results Serum cardiac troponin T in ISO-treated and EXE rats 5 h (5H) and 24 h (recovery) after the onset of interven
exercise), and corresponding CON. ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. Heart Cell Changes and Blood cTnT after Exercise J. Nie et al. troponin release and histological changes associated with
irreversible cardiomyocyte injury (Acikel et al. 2003). We
confirmed both significant cTnT release and histological
evidence of irreversible cardiomyocyte injury with ISO. The mitochondria from ISO-REC19H samples presented
with cristae and matrix proteins disintegration and/or
accumulation of large dense granules in the matrix in
focal groups of myofibers. Ruptured and fragmented
mitochondria also appeared in some instances histological
scans. Disrupted mitochondria shut down ATP produc-
tion which leads to a state of irreversible injury (Cheville
2009). The
mechanism(s)
of
ISO-induced
injury
to
myocardial cells might involve hypoxia, calcium overload
and excessive production of free radicals (Acikel et al. 2003). In this model, the severity of the lesion in the
myocardium
is
directly
proportional
to
the
dosage
administered (Ferrans et al. 1969). Based on the effects of
different dosages of ISO reported (Ferrans et al. 1969;
Acikel et al. 2003; Goldspink et al. 2004; O’Brien et al. 2006), 5 mg kg1 body weight used in this study is the
minimum dosage to produce irreversible cardiomyocyte
injury in rats. Figure 2. Electron micrograph of myocardium from a control rat
showing intact mitochondria. There is substantial evidence showing that blood levels
of cTnT closely parallel the severity of myocardial injury
(Collinson et al. 2003). This may explain the fact that, in
this study, the median cTnT data following ISO injection
were ~50 times above the response postexercise. The large
difference in serum cTnT release probably reflects the
release of cTnT from different “pools” within the car-
diomyocytes. In ISO-treated rats severe irreversible car-
diomyocyte
injury
allows
all
cellular
compartments
containing cTn to contribute to elevated serum level. Fur-
thermore, the delayed recovery of serum cTnT in ISO-trea-
ted rats is consistent with a continuous liberation of cTnT
from disintegrating myofibrils observed in other similar
ISO-treated animal studies (Goldspink et al. 2004). Table 3. Electron microscopic evaluation of mitochondrial injury
scoring (means SD, per gird). Exercise-induced cTnT release and
ultrastructural changes The release of cTnT into the blood after prolonged exer-
cise has been described earlier in our animal swimming
models (Nie et al. 2010) as well as numerous human
exercise studies (Shave et al. 2010; Eijsvogels et al. 2016). Middleton et al. (2008) observed cTnT elevations in all
well-trained men both during and after a treadmill mara-
thon using multiple time point measurements. The fact
that cTnT was present in the circulation of all rats at pos-
texercise in this study supports the findings Middleton
et al. (2008) and suggests an almost obligatory response/
phenomenon. Discussion This study demonstrated that cTnT appeared in the circu-
lation of rats after both a single bolus injection of ISO and
5 h of forced swimming. cTnT release was greater without
full recovery at 24 h after ISO compared to exercise. Addi-
tionally, cTnT appearance after ISO was associated with
histological evidence of irreversible cardiomyocyte dam-
age. Uniquely, we observed that the cTnT appearance after
5 h of exercise in rats was not associated with histological
evidence of irreversible cardiomyocyte injury. Results Groups
Severity scores
Extent scores
ISO-REC19H (n = 8)
3.31 0.35
3.16 0.33
EXE-REC19H (n = 8)
0.18 0.111
0.18 0.111
SAL-REC19H (n = 8)
0.11 0.061
0.11 0.061
ISO, isoprenaline; EXE, exercise; SAL, saline. 1Significantly different from corresponding ISO-REC19H value,
P < 0.05. Table 3. Electron microscopic evaluation of mitochondrial injury
scoring (means SD, per gird). ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. Effects of bolus ISO injection on serum cTnT
and histological evidence of cardiomyocyte
damage Experimental injection of ISO in animals is a well-estab-
lished model to study the relationship between cardiac The current data are the first to assess the association
of
exercise-induced
circulating
cTnT
elevation
with 2016 | Vol. 4 | Iss. 24 | e13083
Page 5 e13083
Page 5 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. J. Nie et al. Heart Cell Changes and Blood cTnT after Exercise reported no blood cTnT data (Maher et al. 1972). A small
of fraction of mitochondria appeared swollen in exercise
and control scans. Mitochondria are exquisitely sensitive
to changes in homeostasis and the swelling that develops
after stress may reflect the entry of water into the mito-
chondrial matrix (Cheville 2009). No differences in mito-
chondrial appearance were noted between the exercise
and control group. That fact that exercised rats displayed a lower cTn
response compared to ISO rats seems to support the the-
ory that only cytosolic cTnT (5% of total cTnT) “leak”
were available to leak into the blood stream (Shave et al. 2010). The resolution of electron microscopy cannot
identify small changes in membrane permeability and as
such we cannot confirm the precise source of blood cTnT
in exercise rats, and this requires further study. It is important to note that the current conclusion is at
odds with Chen et al. (2000) and Olah et al. (2015). Both
studies noted that swimming exercise in rats resulted in
histological evidence of myocarditis-like changes in car-
diac structure. Whether this simply reflects prolonged
exercise-induced immunosuppression with a presumed
increased susceptibility for infection (Harris 2011) is not
known. Based on the light microscopic data alone, how-
ever, it is difficult to draw any definitive conclusion on
the nature of cardiomyocyte damage in these studies (re-
versible or irreversible) (Ishikawa et al. 1997; Chen et al. 2000). Figure 3. Electron micrograph of myocardium from an
isoproterenol-treated rat showing disrupted mitochondria (arrow). Figure 4. Electron micrograph of myocardium from an exercise rat
showing intact mitochondia, but presence of mitochondrial
aggregation (arrow). Implications This study supports the notion that a postexercise cTn
increase represents a physiological process that may
include reversible changes in cardiomyocyte ultrastructure
(George et al. 2011). The cTn elevations are modest and
kinetic data suggest a rapid return to normal. In this
regard, our data suggest that a cTnT response postexercise
is common, if not obligatory, but is unlikely to reflect an
irreversible insult to the cardiomycoytes in the absence of
other elements of clinical disease or risk. In addition, a reduction in cardiac function subsequent
to prolonged exercise in healthy humans has been widely
reported (Oxborough et al. 2010), which raise the ques-
tion of whether acute prolonged exercise could produce
persistent myocardial dysfunction (Shave et al. 2010). Our current findings do not support the possibility, at
least on the cardiomyocytes basis. Nevertheless, care is
still required when interpreting cTn data in patient assess-
ment after significant exercise exposure. What is still not
known is whether the highest cTnT values detected in
human work (Nie et al. 2011b) represent a threshold for
differentiating between reversible and irreversible myocar-
dial changes in response to exercise. Figure 4. Electron micrograph of myocardium from an exercise rat
showing intact mitochondia, but presence of mitochondrial
aggregation (arrow). histological changes in myocardium via electron micro-
scopy. Our results demonstrated that no cardiomyocytes
exhibited cristolyis and more severe mitochondrial injury
in exercised rats. These findings are consistent with those
of another early study, which used an exhaustive treadmill
running model in rats (exercise duration: ~3 h) but 2016 | Vol. 4 | Iss. 24 | e13083
Page 6 Heart Cell Changes and Blood cTnT after Exercise J. Nie et al. J. Nie et al. extreme
environmental
conditions
including
hypoxia
and/or high temperature. Third, the lack of parallel car-
diovascular dynamics measurements limits further inter-
pretation of the current findings, though the reduction
in cardiac function subsequent to prolonged exercise has
been described in numerous human exercise studies
(Oxborough et al. 2010) as well as animal swimming
models
(Olah
et al. 2015). Fourth,
cTnI
was
not
included
in
this
study. Although
our
prior
studies
observed a high-positive correlation between post-exer-
cise cTnT and cTnI values (Nie et al. 2008, 2011b),
additional cTnI assessment is recommended for future
research, given the potentially higher myocardial speci-
ficity of cTnI (Mair 1997). Implications Finally, recently there is
increasing evidence that intense endurance activity may
particularly tax the right ventricle (RV), which seems
more vulnerable to exercise-induced damage or fatigue,
as compared with the LV (La Gerche 2013). Therefore,
additional RV assessment is recommended for future
research. Recently, Olah et al. (2016) showed differences in
energy-dependent early diastolic function and mechano-
energetics between long-term exercise training- (physio-
logical) and pressure overload-induced (pathological) left
ventricular hypertrophy could be explained by alterations
in mitochondrial regulation, as reflected by normal mito-
chondrial
biogenesis
in
physiological
hypertrophy,
whereas
a
marked
mitochondrial
dysfunction
was
observed in pathological hypertrophy. In this study, we
support and extend the training work (Olah et al. 2016)
by demonstrating the acute effects of intense exercise on
mitochondria in heart. Specifically, the mitochondrial
appearance of exercise rats was normal, and was similar
to that of control rats. The results from this study, along
with the Olah et al. (2016) work, seem to suggest that the
differences between physiological (exercise) and patholog-
ical cardiac hypertrophy are doomed from the start. Moreover, recent evidence also showed that mechano-
energetics recovery contributes to myocardial reverse
remodeling with reductions in load (Ruppert et al. 2016). Although myocardial adaptations to left ventricular assist
devices (LVAD) support indicate substantial potential for
structural, functional, and molecular plasticity within
severely diseased hearts, sustained myocardial recovery
after LVAD removal has been reported in only a small
percent of cases, although higher rates may be possible
(Hellawell and Margulies 2012). It was presumed that the
loss of excessive myocardium to necrosis was important
contributing factor; however, the validated evaluation
index is lack (Hellawell and Margulies 2012). The acute
differences in mitochondrial appearance observed in this
study may serve as a starting point for future reverse
remodeling studies. Conclusions In summary, data from this study confirm that rats
injected with a single bolus of ISO demonstrate sub-
stantial and sustained release of cTnT that is associated
with severe and irreversible histological evidence of car-
diomyocyte injury. Exposure of the animals to a sub-
stantial exercise stimulus resulted in cTnT appearance in
all animals although the magnitude was blunted and
recovery to baseline by 24 h had occurred. Importantly
this study demonstrated that the elevation of cTnT pos-
texercise was not associated with any electron micro-
scopy
based
histological
evidence
of
irreversible
cardiomyocyte injury. We suggest that exercise-induced
changes in serum cTnT may be an obligatory physiolog-
ical response but is not associated with irreversible car-
diomyocyte damage. ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society. Limitations and future research While our study provides novel insight into the nature
of cardiomyocyte damage and cTnT release with exer-
cise, several limitations should be considered and future
studies are warranted. First, this is a single acute study
with short recovery observation period and does not
reflect the nature of exposure in a trained myocardium,
such as in athletes completing lifelong endurance train-
ing, and will require prospective longitudinal studies for
confirmation. Second, in this study, rats in the exercise
groups were forced to swim for 5 h with 5% body
weight attached to their tail. This model cannot be
directly translated to humans although it does represent
a significant physiological challenge to the animal. Our
results cannot be directly extrapolated to the range of
exercise
undertaken
by
humans,
which
may
involve
repeated periods of demanding physiological stress with
limited
recovery,
and
probably
in
conjunction
with Acikel, M., M. E. Buyukokuroglu, H. Aksoy, F. Erdogan, and
M. K. Erol. 2003. Protective effects of melatonin against
myocardial injury induced by isoproterenol in rats. J. Pineal
Res. 35:75–79.
Chen, Y., R. C. Serfass, S. M. Mackey-Bojack, K. L. Kelly, J.
L. Titus, and F. S. Apple. 2000. Cardiac troponin T
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prolonged intense exercise. J. Appl. Physiol. (1985)
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M. K. Erol. 2003. Protective effects of melatonin against
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Hybridization of long short-term memory neural network in fractional time series modeling of inflation
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TYPE Methods
PUBLISHED 04 January 2024
DOI 10.3389/fdata.2023.1282541 TYPE Methods
PUBLISHED 04 January 2024
DOI 10.3389/fdata.2023.1282541 TYPE Methods
PUBLISHED 04 January 2024
DOI 10.3389/fdata.2023.1282541 KEYWORDS
inflation rate, ARFIMA, heteroscedasticity, ARFIMA-GARCH, ARFIMA-LSTM COPYRIGHT © 2024 Arif, Herlinawati, Devianto, Yollanda
and Permana. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is
permitted, provided the original author(s) and
the copyright owner(s) are credited and that
the original publication in this journal is cited, in
accordance with accepted academic practice. No use, distribution or reproduction is
permitted which does not comply with these
terms. KEYWORDS
inflation rate, ARFIMA, heteroscedasticity, ARFIMA-GARCH, ARFIMA-LSTM OPEN ACCESS EDITED BY
Feng Chen,
Dallas County, United States
REVIEWED BY
Jinran Wu,
Australian Catholic University, Australia
Xujiang Zhao,
NEC Laboratories America Inc., United States
*CORRESPONDENCE
Dodi Devianto
ddevianto@sci.unand.ac.id
RECEIVED 24 August 2023
ACCEPTED 06 December 2023
PUBLISHED 04 January 2024
CITATION
Arif E, Herlinawati E, Devianto D, Yollanda M
and Permana D (2024) Hybridization of long
short-term memory neural network in
fractional time series modeling of inflation. Front. Big Data 6:1282541. doi: 10.3389/fdata.2023.1282541 EDITED BY
Feng Chen,
Dallas County, United States
REVIEWED BY
Jinran Wu,
Australian Catholic University, Australia
Xujiang Zhao,
NEC Laboratories America Inc., United States Erman Arif1, Elin Herlinawati2, Dodi Devianto3*, Mutia Yollanda3
and Dony Permana4 1Information system study program, Universitas Terbuka, Tangerang Selatan, Indonesia, 2Mathematics
study program, Universitas Terbuka, Tangerang Selatan, Indonesia, 3Department of Mathematics and
Data Science, Universitas Andalas, Padang, Indonesia, 4Department of Statistics, Universitas Negeri
Padang, Padang, Indonesia Arif E, Herlinawati E, Devianto D, Yollanda M
and Permana D (2024) Hybridization of long
short-term memory neural network in
fractional time series modeling of inflation. Front. Big Data 6:1282541. Inflation
is
capable
of
significantly
impacting
monetary
policy,
thereby
emphasizing the need for accurate forecasts to guide decisions aimed at
stabilizing inflation rates. Given the significant relationship between inflation and
monetary, it becomes feasible to detect long-memory patterns within the data. To capture these long-memory patterns, Autoregressive Fractionally Moving
Average (ARFIMA) was developed as a valuable tool in data mining. Due to the
challenges posed in residual assumptions, time series model has to be developed
to address heteroscedasticity. Consequently, the implementation of a suitable
model was imperative to rectify this efect within the residual ARFIMA. In this
context, a novel hybrid model was proposed, with Generalized Autoregressive
Conditional Heteroscedasticity (GARCH) being replaced by Long Short-Term
Memory (LSTM) neural network. The network was used as iterative model to
address this issue and achieve optimal parameters. Through a sensitivity analysis
using mean absolute percentage error (MAPE), mean squared error (MSE), and
mean absolute error (MAE), the performance of ARFIMA, ARFIMA-GARCH, and
ARFIMA-LSTM models was assessed. The results showed that ARFIMA-LSTM
excelled in simulating the inflation rate. This provided further evidence that
inflation data showed characteristics of long memory, and the accuracy of the
model was improved by integrating LSTM neural network. 1 Introduction Big data are datasets of huge size or complexity, beyond the capabilities of conventional
data-processing applications. While data with numerous entries holds higher statistical
power, those with more attributes or columns can exhibit a larger false discovery rate. The term “big data” is now commonly used to describe the application of advanced
data analytics models to massive data, rather than referring to a specific quantity. These
models consist of predictive analytics, user behavior analysis, and other models that extract
value from big data. In numerous fields including Internet searches, fintech, healthcare
analytics, geographic information systems, urban informatics, and business informatics,
massive datasets consistently pose challenges for scientists, corporate executives, medical
practitioners, advertisers, and government officials. Frontiers in Big Data Frontiers in Big Data 01 frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 in precise short-term forecasting and often indicates extended flat
modeling periods. Inflation refers to a data mining phenomenon that poses
economic challenges for both governments and citizens in
developing nations, including Indonesia. The issue of inflation
represents a crucial indicator in preserving the stability of an
economy (DeLong, 1997). According to Bank Indonesia, inflation
can be defined as a general and sustained upsurge in the prices
of goods and services over a specific period. It is important to
note that an increase in the price of one or two goods alone
does not constitute inflation, except this increase extends to
other goods. When inflation escalates persistently and without
control, it transforms into hyperinflation. The repercussions of
hyperinflation reverberate negatively in the economic growth of
a nation and bear an impact on the socioeconomic conditions of
the populace. Additionally, this phenomenon complicates decision-
making for economic stakeholders in taking the next step. The
situation becomes further intricate when the domestic inflation
rate surpasses those of other countries, which subsequently exerts
pressure on the depreciating rupiah value. However, excessively
low inflation also raises concerns, as prolonged low inflation
can signal an economy operating below its potential capacity,
leading to decreased economic growth and limiting the scope of
monetary policies designed to bolster the economy. Inflation can
be stated to be short-term and long-term, hence, it necessitates
both anticipation and action to avert extreme highs and lows. The key to maintaining inflation within manageable bounds lies
in the formulation of economic policies aimed at inflation control
(Rahman et al., 2020). In some instances, time series data exhibit robust correlations
over a prolonged observation period. 1 Introduction This is evident when
autocorrelation values in the Autocorrelation Function (ACF)
plot decline gradually over an extended span. In this aspect,
the differencing value (d) becomes a real number, which is
addressed by the Auto-regressive Fractionally Integrated Moving
Average (ARFIMA) model (Huang et al., 2022). In 1980, Granger
and Joyeux introduced ARFIMA model, an advancement of
ARIMA model capable of predicting both short and long-memory-
patterned time series data. This flexibility extends to the ability
of ARFIMA to predict data exhibiting short-term as well as long-
term memory patterns (Devianto et al., 2022). Subsequently, in
1981, Hosking examined the properties of long memory patterns in
both stationary and non-stationary ARFIMA models. The Geweke
and Porter-Hudak (GPH) model, which directly determines the
fractional order value of differencing (d) without prior specification
of significant AR and MA orders, offers a specific model for defining
the fractional order value of differencing (d). In the context of time series modeling, the application of
the model hinges on the fulfillment of certain assumptions for
the residuals. These assumptions entail non-autocorrelation,
non-heteroscedasticity,
and
normality. Heteroscedasticity,
a
phenomenon in the model, typically arises due to the random
fluctuations in the data, leading to fluctuating variance within the
model. Addressing this issue necessitates an advanced strategy,
as the variance in the model continues to fluctuate randomly. To
address this challenge, the classical time series model known as
the Generalized Autoregressive Conditional Heteroscedasticity
(GARCH) is employed. However, it remains crucial to uphold
the residual assumption. This model assists in estimating optimal
parameters for nonlinear models through alternative iterative
processes, resulting in accurate approximations of real values. A nonlinear model involves employing numerical or iterative
processing on extensive data, enabling precise approximation
of actual values. Among the popular nonlinear models, the
neural network stands out. The neural network employs iterative
processing, starting with random weight adjustments, followed
by subsequent modifications to enhance initial weights (Yollanda
et al., 2018). During the training of substantial data, initial weights
are refined through the backpropagation model to correct errors. This model is combined with classical time series using the integer
order model (ARIMA) and applied to counterbalance the residual
of ARIMA model, which includes the heteroscedasticity effect
(Devianto et al., 2023). In the domain of finance, the fractional
order of ARIMA, known as ARFIMA, anticipates abrupt stochastic
fluctuations within financial markets. Frontiers in Big Data frontiersin.org 2.1 Data source The study included datasets of 162 monthly inflation records,
spanning from February 2009 to July 2022. The data source
was available on the Bank Indonesia website, specifically at
https://www.bi.go.id. ADF =
ˆδ
SE(ˆδ)
(2) (2) for ˆδ as the estimated δ which is obtained by using ordinary least
squares and SE(ˆδ) as the standard error of δ. The initial hypothesis
is δ = 0, which means that the data is not stationary. The criteria
for decision-making reject the initial hypothesis if the ADF value is
less than the test statistics in the table. 1 Introduction In addition, it integrates
the dynamic aspects of deep learning through Long-Short-Term
Memory (LSTM) network (Bukhari et al., 2020). By considering various factors in monetary policy, Bank
Indonesia typically adjusts the benchmark interest rate in response
to projected deviations from the established inflation target. To
facilitate this forecast, predictions of inflation patterns play a vital
role in preempting unstable macroeconomic conditions (Hasenzagl
et al., 2022). These predictions often draw on mathematical
sciences, employing time series models to analyze the movement of
inflation data. Time series models arrange data chronologically and
leverage the presumed repetition of patterns from past periods into
the present and future. The purpose of time series model analysis
is to uncover patterns for modeling future events (Alyousifiet al.,
2021), identifying a variety of patterns that show to be influential
on the response variable (Wu et al., 2023), enhancing forecasting
accuracy and stability from the perspectives of noise distribution
and outliers (Yang et al., 2023), and exploring how a particular
model selection can be better applied to forecast the new data in
the future (Xu et al., 2023). Based on the abundant data and its
diverse attributes consisting of trends, seasonality, and cyclicality,
current values are often modeled based on past data exhibiting
inter-variable correlations, commonly through linear or nonlinear
models. Time series data patterns are typically categorized into short-
term and long-term patterns. Short-memory patterned time series
data feature weak correlations within short periods. The Auto-
Regressive Integrated Moving Average (ARIMA) model serves to
capture these patterns, requiring data to be stationary. Stationarity
is achieved through differencing, where the differencing value (d)
is a non-negative integer. The ARMA model, which is a fusion
of the AR and MA models, combines autoregressive variables and
past residues to model variable movement. ARIMA model excels LSTM model, belonging to the category of recurrent neural
network, effectively overcomes the challenge of vanishing gradients
that often arise in training. Its versatility allows for application
across various topics, including the intricate and volatile financial
market (Gajamannage et al., 2023), and specific cases such as asset
pricing in the Chinese stock market (Pan et al., 2023), due to
its ability to mitigate error propagation during iterations. Neural Frontiers in Big Data 02 frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 • Cyclicality: Implies the presence of long-term tendencies such
as economic booms and busts. 1 Introduction • Cyclicality: Implies the presence of long-term tendencies such
as economic booms and busts. network also finds usefulness in predicting energy demand and
CO2 emissions (Javanmard et al., 2023). In this study, a fusion
of a multiobjective mathematical model with data-driven machine
learning algorithms enhances the accuracy of energy demand and
CO2 emissions forecasts in the transportation sector. • Irregularity: Accounts for random or unforeseen fluctuations
in data. • Exogenous
factors:
Entail
external
influences,
namely
monetary policies, crises, or societal shifts. The implications for employing a statistical procedure, neural,
and combined techniques for time-series forecasting of findings in
the healthcare domain to forecast the expenditures of two different
pain medications are demonstrated by combining ARIMA, neural
network, and LSTM with the integer value of differencing
(Kaushik et al., 2020). According to the studies related study, the
suggested model requires to be verified by evaluating the residual
assumption using a classical time series autoregressive moving
average with either an integer or fractional differencing order. Since the inflation data fluctuates randomly over time, the model
typically exhibits heteroscedasticity. This study makes use of a
long short-term memory neural network (LSTM) and generalized
autoregressive conditional heteroscedasticity (GARCH) to provide
some alternative suggested techniques to improve the sensitivity
of heteroscedasticity effects present in residual classical time series
data with long-memory patterns. To create a time series model incorporating long memory
elements, ARFIMA model follows a series of stages: Step 1. Checking the stationarity of the data with respect to
variance. Let {Xt} be a time series data sequence. Since the data
are not stationary, data transformation was performed to obtain a
rounded value (λ) using Box-Cox transformation T. For example,
assuming data Xt is non-stationary regarding variance, it can be
transformed with the formula T(Xt) = (Xλ−1
t
)/λ, where λ is the
transformation parameter. Stationarity is achieved when λ = 1
yields a rounded value process (Devianto et al., 2022). Step 2. Checking the stationarity of the data with respect to
mean by using Augmented Dickey-Fuller (ADF) test. The random
walk equation with drift for the differenced-lag model is regressed
to be: ∇Xt = µ + δXt−1 +
k
X
i=1
φi∇Xt−i + et
(1) (1) 2 Materials and methods for ∇Xt = Xt −Xt−1, k is the number of lags, δ is the slope
coefficient, µ is a drift parameter, φi is parameter of random walk
equation, and et is white noise error term. The test statistic is used
as follows: for ∇Xt = Xt −Xt−1, k is the number of lags, δ is the slope
coefficient, µ is a drift parameter, φi is parameter of random walk
equation, and et is white noise error term. The test statistic is used
as follows: frontiersin.org 2.2 Long-memory time series of
autoregressive fractional integrated
moving average 10.3389/fdata.2023.1282541 determination coefficient value R2 and the sample size n, expressed
as follows: Step 7. Estimating parameters and testing the significance
of ARFIMA model. Parameter estimation is performed on each
model, followed by significance tests. A model is considered feasible
when its parameters are significant, with probability values smaller
than α = 5%. LM = nR2
(6) (6) Step 8. Selecting the best ARFIMA model based on the smallest
Information Criterion (AIC) value of Akaike. The LM test followed a chi-squared distribution with degrees
of freedom equal to k −1, where k was the number of estimated
parameters. The initial hypothesis was rejected because the statistic
value was greater than the critical value LM > χ2
α(k −1). However,
the hypothesis could be rejected when the probability value was less
than the significance level. Step 9. Testing the residual assumptions of the best ARFIMA
model, including non-autocorrelation and normality. Step 10. Determining the best ARFIMA model equation and its
interpretation. A time series Xt is classified as a sequence of white noise
when an uncorrelated random variable, adhering to a specific
distribution, maintains a constant mean of zero and a constant
variance of Var(Xt) = sigma2 along with Cov(Xt+h, Xt) = 0
for k ̸= 0. Among classical time series, ARIMA combines AR
and MA models after integer differencing. ARIMA evolved into
ARFIMA, which integrates ARFIMA features. ARFIMA parallels
the structure of ARIMA but relies on fractional values for
differencing, as opposed to ARIMA integer differencing. Let φp(B)
as AR components, θq(B) as MA components, B as the operator of
backward shift, and (1 −B)dXt indicates the d-order differenced
stationary time series, the process is labeled ARFIMA(p, d, q)
(Devianto et al., 2023): The final assumption was the normality test, where the initial
hypothesis stated that the residual skewness (S) and kurtosis (K)
of ARFIMA model matched a normal distribution with expected
values of zero. This was determined using the Jarque-Bera (JB) test,
and the statistics test of JB could be expressed as follows (Devianto
et al., 2023): JB = n
6
S2 + (K −3)2
4
(7) (7) where K and S are kurtosis and skewness, respectively. The
initial hypothesis was rejected since the statistic value was greater
than the critical value JB > χ2
α(2) or when the probability value
was less than the significance level of 5%. 2.2 Long-memory time series of
autoregressive fractional integrated
moving average φp(B)(1 −B)dXt = θq(B)εt
(4) When fitting ARFIMA model, potentially significant models
were selected, and the best model was selected. The goodness-
of-fit criteria such as AIC, BIC, and HQ were applied, using
the loglikelihood function to determine the best model. Let ˆσ 2
ε
represent the maximum likelihood estimator of σ 2
ε , k denoted
the number of estimated parameters, and n indicated the number
of observations. The equations for AIC, BIC, and HQ were
systematically written as follows: φp(B)(1 −B)dXt = θq(B)εt (4) with with φp(B) = (1 −φ1B −φ2B2 −· · · −φpBp)
θq(B) = (1 −θ1B −θ2B2 −· · · −θqBq) where p, q, and B are the positive integer values. AIC = n ln( ˆσε2) + 2k
(8)
BIC = n ln( ˆσε2) + kln(n)
(9)
HQ = n ln( ˆσε2) + 2kln(ln(n))
(10) ARFIMA model that had been fitted was then extended into
time series analysis to determine whether the residual assumptions
were met. It should be noted that these assumptions began with
autocorrelation and further consisted of heteroscedasticity and
normality. The initial hypothesis assumed that there was no linear
relationship in dependency within the residual ARFIMA model. The statistic value QLB could be expressed as follows (Devianto
et al., 2023): (10) The best model was selected based on the smallest value among
AIC, BIC, and HQ. Assuming there were two smallest values in any
of these criteria, a nonparametric model was required to determine
the smallest rank. QLB = n(n + 2)
k
X
i=1
ρ2
i
n −i
(5) (5) Frontiers in Big Data 2.2 Long-memory time series of
autoregressive fractional integrated
moving average Step 3. As graphically, plotting the autocorrelation function
(ACF) of the transformed data in Step 1 to detect the presence of
a long-memory effect. If the ACF pattern is generated as a sine
function, there is a long-memory pattern data. As mathematically,
long-memory pattern data can be used the differencing parameter
d explained in Step 4. Managing big data required employing numerical processing
to identify data patterns and approximate optimal parameters. Various statistical models were used to address fluctuations rooted
in data patterns. An example of this model was time series analysis,
used to scrutinize data mined over time, often involving significant
data volumes. This model aimed to forecast future events and
comprehend underlying processes by studying data patterns and
trends. Time series analysis found applications in diverse fields,
including forecasting consumer demand, predicting stock market
shifts, and understanding economic indicators such as inflation and
unemployment trends. These models assisted in comprehending
trends, seasonality, and noise within data, using past data to predict
future values. Furthermore, time series analysis could predict the
impact of exogenous factors, namely governmental policy changes
or technological advancements. Various elements could have an
impact on a time series (Gulmez, 2023): Step 4. Estimating the differentiating parameters d using the
Geweke and Porter-Hudak model, denoted as bdGPH with the
following formula (Devianto et al., 2022): bdGPH =
Pm
j=1(xj −¯x)(yj −¯y)
Pm
j=1(xj −¯x)2
(3) (3) where yj = lnI(λj) and xj = −ln[2sin( λj
2 )]2. The I(λj) function
is a periodogram with a frequency of Fourier λj =
2πj
T , j =
1, 2, · · · , m, and T is the number of observation data while m is the
limit of the number of Fourier frequencies. Step 5. Transforming the differential data using the obtained
bdGPH values. • Trend: Refers to an entire direction, whether ascending,
descending, or stable, that a time series displays over time. • Trend: Refers to an entire direction, whether ascending,
descending, or stable, that a time series displays over time. Step 6. Identifying potential ARFIMA model by combining
significant orders of autoregressive (AR) and moving average (MA)
models using PACF and ACF plots of stationary data, respectively. • Seasonality: Denotes recurring trends within data, such as
heightened sales during holidays. • Seasonality: Denotes recurring trends within data, such as
heightened sales during holidays. Frontiers in Big Data 03 frontiersin.org frontiersin.org Arif et al. frontiersin.org 2.3 Improving volatility sensitivity with
classical generalized autoregressive
conditional heteroschedasticity where n is the number of data, the sample auto-correlation
coefficient at lag k = 1, 2, 3, · · · , K is denoted as ρ2
i , and lag length is
denoted as K. The initial hypothesis was rejected when the statistic
value was greater than the critical value or QLB > χ2
α(k −p −q). On the other hand, the hypothesis could be rejected assuming the
probability value was less than the significance level. Time-series analysis involved four factors, namely trend,
seasonality, periodicity, or cycle, as well as irregular components,
all of which generally contributed to the variations in the data. In
1982, Engle introduced ARCH, assuming that the data variance
was influenced by past values. Let Q denote the number of
autoregressive terms in the model, t represents the ordinary least
squares variance obtained from the original regression model
Equation (4), αi stands for a parameter of ARCH, εt = σtet, and
et ∼N(0, 1) with αi > 0. The variance assumption of ARCH and The second assumption concerned the heteroscedasticity effect
using the Lagrange Multiplier (LM) test by White. The initial
hypothesis of the LM test assumed that the residual ARFIMA model
exhibited homoscedasticity, where the variance of this residual
model remained constant, allowing the random fluctuated data to
be ignored. The statistic value of the LM test was the product of the 04 frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 ARIMA in Equation (4) allowed ARCH(Q) to be stated as follows
(Devianto et al., 2023): as follows: zj = f (inj) = f
n
X
i=0
xiwij
! (13) (13) σ 2
t = α0 + α1ε2
t−1 + · · · + αQε2
t−Q
(11) (11) This result was then passed as input to other neurons and
the activation function f scaled the output zj into appropriate
ranges. The network architecture could be classified as feed-forward
(FFNN) and recurrent neural network (RNN), commonly used for
forecasting. In a feed-forward network, each input unit received
data from the layer below. Meanwhile, the outputs of a recurrent
network became inputs in the preceding layer. The recurrent
network created a dynamical system where inputs depended on the
initial values of previous inputs, in order to simulate a stable state
(Devianto et al., 2023). for i = 0, 1, 2, · · · , Q. 2.3 Improving volatility sensitivity with
classical generalized autoregressive
conditional heteroschedasticity As further, the following steps show how to design
the Long Short-Term Memory (LSTM) neural networks: In constructing the structural LSTM model, LSTM neural
network comprised input gates, memory cells, forget gates, and
output gates. These components processed information over
longer periods. The network could selectively store and retrieve
information as needed, regulated by these gates that controlled
the flow of data into and out of memory cells. The input gate
introduced new inputs to the cell, the forget gate maintained values
for later use, and the output gate determined the output of the cell. Common tasks for which LSTM were employed included language
translation, speech recognition, and stock price prediction. While
RNN could learn long-term dependencies in data, it was impacted
by the vanishing gradient problem. LSTM addressed this issue by
using a set of gates to determine the data to retain and those
to discard. This allowed the model to retain more information
compared to RNN, achieved through gradient control (Haider
et al., 2019). A typical LSTM cell structure could be seen in Figure 2. 2.3 Improving volatility sensitivity with
classical generalized autoregressive
conditional heteroschedasticity While processing real data, ARCH often
produced higher orders, leading to an increase in the number
of estimated parameters. In order to replace GARCH model as
the preferred one, ARCH model was created. The residual et was
assumed to be homoscedastic since it represented the concept of
a time series, i.e., E(e2
t ) = E(e2
t |e2
t−1, e2
t−2, · · · ) = σ 2 for each
t. However, the variance of et fluctuated over time in GARCH
model, resulting in E(e2
t |e2
t−1, e2
t−2, · · · ) = σ 2
t , which indicated
heteroscedasticity. GARCH model required that the initialized data had constant
variances. In simpler terms, the fluctuations in the data did not
always have the same value at time t. An et process was considered
a GARCH(P,Q) process when it satisfied the following conditions: The feed-forward neural networks (FFNN) require one or more
input unit(s) in processing in the hidden layer so that the results
will have the output unit. The output unit is then compared to
the observational or actual data as the supervised learning. In the
time series case, there is no explanation which variable that will
be the input or output units so that the time series model have
separated one dataset to be two or more dataset by using the
significant lag in the autocorrelation functions. Therefore, there is
one type of recurrent neural networks that can be used to process
the time series model. A common type of recurrent neural network
was LSTM, designed for analyzing time series data. LSTM was
equipped to handle long-term dependencies present in time series
data, ensuring outcomes depended on previous data values. The
LSTM model has the advantage of processing and predicting time
series events with long intervals and delays. It means that scientists
can use previous data to forecast new data in a few periods ahead. It limits applications of FFNN to time series models. FFNN has to
receive input unit values before calculating the estimated output
unit. Frontiers in Big Data 2.3 Improving volatility sensitivity with
classical generalized autoregressive
conditional heteroschedasticity σ 2
t = α0 +
Q
X
i=1
αiε2
t−i +
P
X
j=1
βjσ 2
t−j
(12) (12) where εt = σtet, et ∼N(0, 1), Q > 0, P ≥0, αi ≥0, α0 > 0,
for i = 0, 1, 2, · · · , Q and βj ≥0 for j = 1, 2, · · · , P and also
PQ
i=1 αi + PP
j=1 βj < 1. The Maximum Likelihood Estimation (MLE) model was then
used to initially approximate GARCH parameters. White noise
with a mean value of 0 and a variance of σ 2 was thought to
make up the residual of ARIMA model. The parameters were
estimated using iterative procedures, such as Newton-Raphson,
after obtaining the log-likelihood function for n observed data
points. This model was used to solve the probability log function. Consequently, an estimator that sufficiently converged for each
parameter was obtained. In constructing the structural LSTM model, LSTM neural
network comprised input gates, memory cells, forget gates, and
output gates. These components processed information over
longer periods. The network could selectively store and retrieve
information as needed, regulated by these gates that controlled
the flow of data into and out of memory cells. The input gate
introduced new inputs to the cell, the forget gate maintained values
for later use, and the output gate determined the output of the cell. Common tasks for which LSTM were employed included language
translation, speech recognition, and stock price prediction. While
RNN could learn long-term dependencies in data, it was impacted
by the vanishing gradient problem. LSTM addressed this issue by
using a set of gates to determine the data to retain and those
to discard. This allowed the model to retain more information
compared to RNN, achieved through gradient control (Haider
et al., 2019). A typical LSTM cell structure could be seen in Figure 2. The mathematical expressions for the inner connections of the
gates in LSTM cell structure were as follows. Let f (t), i(t), c(t), and
o(t) as forget gate, input gate, modulation cell gate, and output gate,
respectively, with their activation functions of σf , σi, σc, and σo. In
the modulation cell gate, the input is updated at the present time
instant. All gates work on current input x(t) and previous values of
states h(t −1). 2.4 Improving volatility sensitivity with
long short-term memory In the context of big data analysis, it was possible to forecast
the future by identifying patterns in past data. Additionally, a
correlation existed between the variables and the historical data
or data residuals. The artificial neural network was a system
that mimicked the network of nerve cells in human and animal
brains (Yollanda et al., 2018). Furthermore, it consisted of a few
hidden layers that connected the input to the output layer. The
connections between two layers were defined by weights and biases,
also referred to as the coefficient of network. The construction of
the mathematical neural network model was generally depicted in
Figure 1 (Devianto et al., 2023). The mathematical expressions for the inner connections of the
gates in LSTM cell structure were as follows. Let f (t), i(t), c(t), and
o(t) as forget gate, input gate, modulation cell gate, and output gate,
respectively, with their activation functions of σf , σi, σc, and σo. In
the modulation cell gate, the input is updated at the present time
instant. All gates work on current input x(t) and previous values of
states h(t −1). As further, the following steps show how to design
the Long Short-Term Memory (LSTM) neural networks: Figure 1 showed the construction of neural network that
connected two layers through directed links of weights or biases to
determine the sign and strength of the input data. The input data xi,
representing x1, x2, · · · , xn was then calculated as a weighted sum of
its inputs on the hidden layer, inj. Each hidden unit j transformed
the input through the activation function f , yielding zj, expressed frontiersin.org 05 Arif et al. 10.3389/fdata.2023.1282541 FIGURE 1
A diagram construction of mathematical neural network model. FIGURE 2
A diagram construction of mathematical long short-term memory neural network model. FIGURE 1
A diagram construction of mathematical neural network model. FIGURE 1
A diagram construction of mathematical neural network model. FIGURE 2
A diagram construction of mathematical long short-term memory neural network model. FIGURE 2
A diagram construction of mathematical long short-term memory neural network model. FIGURE 2
A diagram construction of mathematical long short-term memory neural network model. 1. Load time series dataset X(t) into the networks as the current
input unit. 1. Load time series dataset X(t) into the networks as the current
input unit. a simple method. 2.4 Improving volatility sensitivity with
long short-term memory The networks separated the dataset into 70%
for training the model and 30% for validating the testing data
against the training data. 2. Normalize the dataset x(t). The scale of the input data impacts
LSTMs, especially since the sigmoid (default) or tanh activation
functions are employed. It is recommended to rescale the data to
the 0-to-1 range as new minimum and new maximum dataset,
respectively, also known as normalizing. The normalization of
the dataset can be expressed as follows: 4. Reshape the training and testing data as input units (training
X and testing X) and as output unit (training Y and testing X)
based on the significant lag in autocorrelation function or first
lag as the default. 4. Reshape the training and testing data as input units (training
X and testing X) and as output unit (training Y and testing X)
based on the significant lag in autocorrelation function or first
lag as the default. 5. Design and fit the LSTM neural networks. In this step, the
number of iterations, input unit, hidden layers and their units,
and output units are determined. The default sigmoid activation
function is used for the LSTM blocks or units. 5. Design and fit the LSTM neural networks. In this step, the
number of iterations, input unit, hidden layers and their units,
and output units are determined. The default sigmoid activation
function is used for the LSTM blocks or units. x(t) =
X(t) −min(X(t)))
max(X(t))) −min(X(t)))(new max(X(t)))
−new min(X(t)))) + new min(X(t)))
(14)
=
X(t) −min(X(t)))
max(X(t))) −min(X(t)))(1 −0) + 0
=
X(t) −min(X(t)))
max(X(t))) −min(X(t)))
(15) x(t) =
X(t) −min(X(t)))
max(X(t))) −min(X(t)))(new max(X(t)))
−new min(X(t)))) + new min(X(t)))
(14)
=
X(t) −min(X(t)))
max(X(t))) −min(X(t)))(1 −0) + 0
=
X(t) −min(X(t)))
max(X(t))) −min(X(t)))
(15) (14) 6. Reproduce the optimal weights for each unit. Let Wjh, Wjx, and
Wj0 as the weights for previous hidden unit, input unit, and bias,
respectively for j = f , i, ˜c and o. The forget and input gates are
calculated as follows (Yu et al., 2019): (15) 3. Split the dataset to be training and testing data. The sequence of
values is important since it deals with time series data. You are
able to split the ordered dataset into train and test datasets using 3. Split the dataset to be training and testing data. The sequence of
values is important since it deals with time series data. Frontiers in Big Data frontiersin.org The modulation cell gate value was calculated as: The modulation cell gate value was calculated as: In modeling, evaluation criteria were used to assess how well
it predicted output values from input data (Gulmez, 2023). The
Mean Squared Error (MSE) measured the gap between actual
values and projected values. It was calculated by averaging squared
differences between expected and actual numbers. A lower MSE
value indicated a more accurate model. ˜c(t) = σ˜c
W˜chh(t −1) + W˜cxx(t) + W˜c0
(18)
c(t) = f (t)c(t −1) + i(t)˜c(t)
(19) The current cell state, current input, and previous state of
the cell were used to control the output gate. The updated output
and hidden cell state were given as follows: MSE = SSE
n
=
Pn
t=1(yt −ˆyt)2
n
(22) (22) o(t) = σo
Wohh(t −1) + Woxx(t) + Wo0
(20)
h(t) = o(t)σh(c(t))
(21) The accuracy of a model was quantified by the Mean Absolute
Percentage Error (MAPE), expressed as a percentage. It was
computed by dividing the absolute difference between expected and
actual values by the actual value, then averaging these percentages
and calculating the difference between predicted and actual values. A lower MAPE number indicated better real-world prediction by
the model. (20)
(21) The design of deep network generally involved multiple
hidden layers. To achieve the best results in the proposed
work, several LSTM hidden layer topologies were evaluated. The
optimal weights are obtained after optimizing the function. MAPE = 1
n
N
X
t=1
|yt −ˆyt|
yt
× 100%
(23) (23) 7. Validate the LSTM neural networks model. The LSTM model
is evaluated in this step to determine whether it is proposed
or requires to be improved. If the error of the training data
is greater than the error of the testing data, the processing is
complete. However, iterations are continued if the training data
has a smaller error than the testing data. It is necessary to
determine whether the LSTM model can recognize unexpected
or new data. Another model to measure the gap between expected and actual
values was the Mean Absolute Error (MAE). This was calculated
by finding the absolute difference between expected and actual
numbers, and then taking their average. The MAE statistic also
assessed the model accuracy, with lower MAE values indicating
higher results. 8. Calculate the evaluation model of Mean Squared Error (MSE),
Mean Absolute Percentage Error (MAPE), and Mean Absolute
Error (MAE). 2.4 Improving volatility sensitivity with
long short-term memory You are
able to split the ordered dataset into train and test datasets using f (t) = σf
Wfhh(t −1) + Wfxx(t) + Wf 0
(16)
i(t) = σi
Wihh(t −1) + Wixx(t) + Wi0
(17) frontiersin.org Frontiers in Big Data
0 frontiersin.org Frontiers in Big Data 06 Arif et al. 10.3389/fdata.2023.1282541 FIGURE 3
Plot of inflation data that starting from February 2009 until July 2022. The modulation cell gate value was calculated as: MAE = 1
n
N
X
t=1
|yt −ˆyt|
(24) (24) frontiersin.org Frontiers in Big Data 07 frontiersin.org 10.3389/fdata.2023.1282541 Arif et al. TABLE 1 Augmented Dickey-Fuller test. stationary concerning the mean value. This was further supported
by the probability value of 0.1353, which was higher than the 5%
significance level. To determine the order differencing of fractional
or integer, the identification pattern used the Autocorrelation
Function (ACF) to ascertain the presence of long-memory terms
as shown in Figure 4. stationary concerning the mean value. This was further supported
by the probability value of 0.1353, which was higher than the 5%
significance level. To determine the order differencing of fractional
or integer, the identification pattern used the Autocorrelation
Function (ACF) to ascertain the presence of long-memory terms
as shown in Figure 4. Critical value
ADF test
Statistics value
p-value
1% : −3.4722
5% : −2.8799
−2.4232
0.1353
10% : −2.5766
Lag order: 2. Figure 4 showed a gradual decrease in data over time, indicating
the presence of a long-memory pattern and suggesting a fractional
order differencing for the model. Mathematically, differencing with
an order value of d was required to make the data stationary
concerning the mean, estimated using the Geweke Porter-Hudak
(GPH) model. The order model determined by the GPH model was
ˆdGPH = 0.4941. As ˆdGPH = 0.4941 was <0.05, the data exhibited
a long-memory effect and could be modeled with ARFIMA. Subsequently, the order of ARFIMA model was determined by
identifying the number of significant lags in the ACF and PACF
plots, as shown in Figure 5. Lag order: 2. After calculating the optimal model, the ratio of sums of squares
of regression to sums of total squares determined the coefficient R2. This measurement ranged from 0 to 1, and the value of R2 close to 0
indicated the estimated model did not fit well, while a value close to
1, implied it was well-fit. Let y be the mean of the dataset yi, where
i = 1, 2, · · · , n. R2 was calculated as follows: R2 =
Pn
i=1( ˆyi −¯y)2
Pn
i=1(yi −¯y)2
(25) Based on Figure 5, the ACF coefficient reached a significant
value at a lag of 5, while the PACF coefficient reached a significant
value at a lag of 2. 3 Main results Table 2
showed
that
the
models
ARFIMA(0, 0.4941, 1),
ARFIMA(0, 0.4941, 2),
ARFIMA(0, 0.4941, 3),
ARFIMA
(0, 0.4941, 4),
ARFIMA(1, 0.4941, 0),
ARFIMA(1, 0.4941, 1),
and ARFIMA(1, 0.4941, 3) were significant and suitable for
building an ARFIMA model. However, not all significant models
were applied in the subsequent steps. In order to identify the
optimal model, a comparison was made between the AIC and
BIC values. The evaluation of these values in Table 2 for the
seven models revealed that the ARFIMA(1, 0.4941, 1) showed
the lowest AIC and BIC values among the available alternatives. Consequently, it can be stated that the ARFIMA(1, 0.4941, 1)
appeared as the most favorable choice. Table 2
showed
that
the
models
ARFIMA(0, 0.4941, 1),
ARFIMA(0, 0.4941, 2),
ARFIMA(0, 0.4941, 3),
ARFIMA
(0, 0.4941, 4),
ARFIMA(1, 0.4941, 0),
ARFIMA(1, 0.4941, 1),
and ARFIMA(1, 0.4941, 3) were significant and suitable for
building an ARFIMA model. However, not all significant models
were applied in the subsequent steps. In order to identify the
optimal model, a comparison was made between the AIC and
BIC values. The evaluation of these values in Table 2 for the
seven models revealed that the ARFIMA(1, 0.4941, 1) showed
the lowest AIC and BIC values among the available alternatives. Consequently, it can be stated that the ARFIMA(1, 0.4941, 1)
appeared as the most favorable choice. This study used 162 data points of inflation in Indonesia on
a monthly basis. This section involved building a long-memory
pattern of ARFIMA model, enhancing volatility residual using
LSTM model, and evaluating the preferred model using certain
criteria. The modulation cell gate value was calculated as: This suggested the possibility of forming an
ARFIMA model by combining a maximum lag of 2 for parameter
p and a maximum lag of 5 for parameter q, along with a dGPH
value of 0.4941. Furthermore, the parameters for each model were
estimated and from the results, a significance test was conducted. The probability values for each model were shown in Table 2. A
model was considered significant when the probability value of its
parameter was <0.05. (25) The value of R2 represented the amount of variance in the
response variable explained by the predictor variable. Furthermore,
it represented the squared correlation between observed values yi
and anticipated values ˆyi based on data processing. Frontiers in Big Data frontiersin.org 3.1 Building long-memory pattern of
ARFIMA model In this subsection, the modeling process was initiated with
the initial step of identifying the movement of inflation data. The
inflation data model with a monthly period was graphically shown
in Figure 3. Relying solely on model selection was insufficient to confirm
that ARFIMA(1, 0.4941, 1) adequately fulfilled the necessary
conditions as a time series. This led to the examination of the
residual assumption of the ARFIMA(1, 0.4941, 1). Table 3 showed
a test of residual assumptions for ARFIMA(1, 0.4941, 1). Based on Figure 3, it could be concluded that the inflation
data pattern for each period exhibited free fluctuations. Since
the inflation data did not fluctuate around a constant mean
and variance, the data were not stationary concerning mean and
variance values. To address this non-stationarity in time series
data regarding variance, data transformation was performed using
the Box-Cox transformation. The first step was determining the
rounded value (λ). Based on the transformation parameter formula,
the value of λ was −14.57178, indicating non-stationarity with
respect to variance. Therefore, a second-stage transformation was
conducted with a λ value of 1, rendering the inflation data
stationary regarding variance. The subsequent step was to check
whether the data were stationary concerning mean values using the
Augmented Dickey-Fuller test, shown in Table 1. Upon reviewing Table 3, it became evident that the p-value
of the autocorrelation test had surpassed 0.05 at the 91st lag,
indicating the absence of correlation among residuals. However, in
the heteroscedasticity and normality tests, the p-values were below
0.05. This suggested the presence of heteroscedasticity or volatility
effects on the residuals, necessitating their adjustment. The
normality test could be overlooked due to the rapid fluctuations
in the time series data. Therefore, ARFIMA(1, 0.4941, 1) was
established as the best model with the following equation: (1 −B)dXt = 81Xt−1 + θ1εt−1 + εt
(1 −B)0.4941Xt = 0.6140Xt−1 + 0.3218εt−1 + εt (1 −B)dXt = 81Xt−1 + θ1εt−1 + εt
(1 −B)0.4941Xt = 0.6140Xt−1 + 0.3218εt−1 + εt From Table 1, the value of the statistic exceeded the critical
value at the 5% significance level, implying that the data were not frontiersin.org frontiersin.org 08 Arif et al. 10.3389/fdata.2023.1282541 FIGURE 4
ACF plot of inflation data exhibiting variance stationarity. FIGURE 5
Plot of autocorrelation and partial autocorrelation function. FIGURE 4
ACF plot of inflation data exhibiting variance stationarity. FIGURE 4
ACF plot of inflation data exhibiting variance stationarity. 3.1 Building long-memory pattern of
ARFIMA model FIGURE 4
ACF plot of inflation data exhibiting variance stationarity. FIGURE 5
Plot of autocorrelation and partial autocorrelation function. FIGURE 5
Plot of autocorrelation and partial autocorrelation function. Frontiers in Big Data
09
frontiersin.org 09 Frontiers in Big Data frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 TABLE 2 The significant estimated parameters of the ARFIMA model with their AIC or BIC value. Model
Estimation parameter
Model selection
Parameter
Estimate
Statistic
Pr(> |z|)
AIC
BIC
ARFIMA(0, 0.4941, 1)
θ1 [MA(1)]
−0.7032
−15.0857
< 2.22e-16
−1653.5420
−1641.1900
ARFIMA(0, 0.4941, 2)
θ1 [MA(1)]
−0.9077
−12.5615
< 2.22e-16
−1675.3520
−1659.9100
θ2 [MA(2)]
−0.3414
−5.4321
6.4070e-08
ARFIMA(0, 0.4941, 3)
θ1 [MA(1)]
−0.9137
−11.5841
< 2.22e-16
−1678.3420
−1659.8200
θ2 [MA(2)]
−0.4605
−5.3404
1.0573e-07
θ3 [MA(3)]
−0.1701
−2.2116
0.0278
ARFIMA(0, 0.4941, 4)
θ1 [MA(1)]
−0.9319
−11.7777
< 2.22e-16
−1681.1090
−1659.5000
θ2 [MA(2)]
−0.5003
−4.7312
2.4497e-06
θ3 [MA(3)]
−0.2863
−3.1530
0.0017
θ4 [MA(4)]
−0.1724
−2.2212
0.0270
ARFIMA (1, 0.4941, 0)
81 [AR(1)]
0.7571
−14.4049
< 2.22e-16
−1681.1800
−1668.8300
ARFIMA(1, 0.4941, 1)
81 [AR(1)]
0.6141
−7.2804
4.5617e-13
−1686.4910
−1671.0500
θ1 [MA(1)]
−0.3212
−3.0764
0.0021
ARFIMA(1, 0.4941, 3)
81 [AR(1)]
−0.8938
−12.1332
< 2.22e-16
−1674.3120
−1652.7000
θ1 [MA(1)]
−1.9758
−17.0232
< 2.22e-16
θ2 [MA(2)]
−1.4138
−9.1893
< 2.22e-16
θ3 [MA(3)]
−0.4371
−5.5881
7.6217e-12 TABLE 3 Residual assumption test of ARFIMA (1, 0.4941, 1) model. TABLE 3 Residual assumption test of ARFIMA (1, 0.4941, 1) model. Residual assumption
Statistic χ2
p-value
Homoscedasticity
11.1380
0.0038
Autocorrelation
[0.1114, 3.8949]
>0.05
Normality
247.6700
<2.2e-16 TABLE 3 Residual assumption test of ARFIMA (1, 0.4941, 1) model. After obtaining ARFIMA, the presence of a heteroscedasticity
effect in the residual ARFIMA prompted the need for an
improved model to address this issue. Sections 3.2 and 3.3
focused on alternative models for addressing heteroscedasticity
effects, including the linear model of Generalized Autoregressive
Conditional Heteroscedasticity (GARCH) and nonlinear model of
Long-Short Term Memory (LSTM). Frontiers in Big Data 3.2 Improving volatility residual ARFIMA
using GARCH Model
Estimation parameter
The best model selection
Parameter
Estimate
z-value
Pr (> |z|)
AIC
BIC
HQ
GARCH(0,1)
β1
0.9880
809.1039
0.0000
−6.9013
−6.8631
−6.8858
GARCH(0,2)
β1
0.4433
10.2208
0.0000
−6.8736
−6.8165
−6.8504
β2
0.5329
12.4415
0.0000
GARCH(1,0)
α1
0.9989
20.9141
0.0000
−7.0468
−7.0087
−7.03137
GARCH(1,1)
α1
0.7335
4.0984
4.1600e-05
−7.0373
−6.9802
−7.0141
β1
0.2655
6.4339
1.2400e-10
GARCH(2,0)
α1
0.9905
5.2035
1.9600e-07
−7.0506
−6.9934
−7.0274
α2
0.0085
1.9832
0.04735
3.3 Improving volatility residual ARFIMA
using LSTM
After
fitting
ARFIMA
to
the
long-patterned
inflation
data
series,
efforts
were
directed
toward
improving
the
heteroscedasticity of the model by addressing the residual. Visual diagnosis could be used to identify the presence of the
heteroscedasticity effect. Outlier data indicated that an advanced
model was necessary to adjust the effect due to data variability. d f
h
d
l h
d
ff
f
resulting from long-term dependencies, even with substantial
data, posed a challenge due to the random fluctuation in residuals. Consequently, the application of LSTM neural network was
deemed necessary (Shewalkar et al., 2019). The original residual of ARFIMA initially applied to inflation
data from February 2009 to July 2022 was shown in Figure 7. This
presentation aimed to provide insights into the fluctuation patterns
of the residual model. Analysis of Figure 7 showed that the residual ARFIMA model
ti
d t
hibit
id
bl
fl
t
ti
i di
ti
th FIGURE 6
The ACF and PACF chart for determining the order of GARCH model. TABLE 4 The significant estimated parameters of the GARCH model with their AIC or BIC value. FIGURE 6
The ACF and PACF chart for determining the order of GARCH model. E 4 The significant estimated parameters of the GARCH model with their AIC or BIC value. TABLE 4 The significant estimated parameters of the GARCH model with their AIC or BIC value. TABLE 4 The significant estimated parameters of the GARCH model with their AIC or BIC value. Model
Estimation parameter
The best model selection
Parameter
Estimate
z-value
Pr (> |z|)
AIC
BIC
HQ
GARCH(0,1)
β1
0.9880
809.1039
0.0000
−6.9013
−6.8631
−6.8858
GARCH(0,2)
β1
0.4433
10.2208
0.0000
−6.8736
−6.8165
−6.8504
β2
0.5329
12.4415
0.0000
GARCH(1,0)
α1
0.9989
20.9141
0.0000
−7.0468
−7.0087
−7.03137
GARCH(1,1)
α1
0.7335
4.0984
4.1600e-05
−7.0373
−6.9802
−7.0141
β1
0.2655
6.4339
1.2400e-10
GARCH(2,0)
α1
0.9905
5.2035
1.9600e-07
−7.0506
−6.9934
−7.0274
α2
0.0085
1.9832
0.04735 Frontiers in Big Data 3.2 Improving volatility residual ARFIMA
using GARCH ACF and PACF charts. Among these combinations, GARCH(0,1),
GARCH(0,2), GARCH(1,0), GARCH(1,1), and GARCH(2,0) had
been relevant parameters for constructing GARCH. Table 4 showed
the estimated parameters for these models, along with their AIC
and BIC values. According to the residual assumptions of ARFIMA(1, 0.4941, 1)
shown in Table 3, the heteroscedasticity assumption had not been
met. Consequently, an advanced model was necessary to enhance
ARFIMA and minimize variance in the residuals. One traditional
time series, GARCH, had been developed to counteract the random
fluctuating variance or heteroscedasticity impact. The creation of
a GARCH involved using ACF and PACF charts to determine the
order of the model. Figure 6 showed the ACF and PACF charts for
GARCH. The optimal model was determined by selecting those with the
lowest AIC or BIC value from among several potentially significant
ARFIMA model. The smallest values for AIC, BIC, and HQ
were identified from Table 4 as GARCH(1, 0), GARCH(2, 0), and
GARCH(1, 0), respectively. Since GARCH(1, 0) had the lowest AIC
and HQ values, it was selected to enhance the residual ARFIMA
model and address the issue of volatility. The residual GARCH(1,
0) ARFIMA(1, 0.4941, 1) model could be expressed as follows: Figure 6 showed the significance of the ACF and PACF
charts at lag 2. The initial conjecture for model orders P
and Q had been combinations of 2 and 0, respectively. Eight
potential models could be constructed using combinations
including GARCH(0,1), GARCH(0,2), GARCH(1,0), GARCH(1,1),
GARCH(1,2), GARCH(2,0), GARCH(2,1), and GARCH(2,2). The
significant parameters had been identified based on the p-value,
which had been lower than the significance level after estimating
parameters for possible models using combinations from the σ 2
t = α1ε2
t−1 + ǫ∗
t = 0.9989ε2
t−1 + ǫ∗
t
(26) (26) where εt = σtet, et ∼N(0, 1), and ǫ∗
t as the residual of the
GARCH model. By combining the ARFIMA and GARCH models,
this new model of ARFIMA(1, 0.4941, 1)-GARCH(1,0) served as a
potential alternative for forecasting future inflation rates. frontiersin.org 10 frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 FIGURE 6
The ACF and PACF chart for determining the order of GARCH model. TABLE 4 The significant estimated parameters of the GARCH model with their AIC or BIC value. 3.3 Improving volatility residual ARFIMA
using LSTM resulting from long-term dependencies, even with substantial
data, posed a challenge due to the random fluctuation in residuals. Consequently, the application of LSTM neural network was
deemed necessary (Shewalkar et al., 2019). After
fitting
ARFIMA
to
the
long-patterned
inflation
data
series,
efforts
were
directed
toward
improving
the
heteroscedasticity of the model by addressing the residual. Visual diagnosis could be used to identify the presence of the
heteroscedasticity effect. Outlier data indicated that an advanced
model was necessary to adjust the effect due to data variability. Modifying the residual heteroscedasticity effect of ARFIMA was
essential. The persistent vanishing/exploding gradient problem The original residual of ARFIMA initially applied to inflation
data from February 2009 to July 2022 was shown in Figure 7. This
presentation aimed to provide insights into the fluctuation patterns
of the residual model. Analysis of Figure 7 showed that the residual ARFIMA model
continued to exhibit considerable fluctuations, indicating the
persistence of volatility in the residual data. However, the residual 11 Frontiers in Big Data frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 FIGURE 7
Historical residual of ARFIMA model. In determining the sensitivity of random initial weights of
LSTM model and parameters of GARCH model, five random
experiments will be executed. The results of five experiments will
be shown in following Table 6. ARFIMA became stationary concerning the mean value, with the
data fluctuating around zero. This was the reason a nonlinear
model was considered for improving the residual data of ARFIMA. The performance level would then be compared with the linear
GARCH discussed earlier. Figure 8 showed the ACF and PACF of
the processed data. Table 6 shows sensitivity random initial weights against the
actual data. Based on Table 6, all five experiments has the values that
approach measures of dispersion actual values: mean, variance, or
standard error. It concludes that LSTM has the best performance to
capture the long-pattern data and fluctuation of the inflation data
because ARFIMA-LSTM has better performance than ARFIMA-
GARCH to approach the measures of dispersion inflation data. The
results also show that the ARFIMA-GARCH doesn’t change for
each experiments since this hybrid model use Newton Raphson to
approach the optimal parameters using high dimensional matrices. The ACF and PACF plots in Figure 8 showed a significant
impact at the twelfth lag. This observation implied that the
inflation in each year was dependent on the value of the next
year. 3.3 Improving volatility residual ARFIMA
using LSTM Consequently, a preferred model aimed at mitigating the
heteroscedasticity effect would involved employing 12 as the
number of significant lags. To enhance the residual of ARFIMA,
LSTM neural network, which was a nonlinear model, would be
applied. This model included training the network on 80% of
the dataset and testing it on the remaining 20% to evaluate
performance. Parameter settings for LSTM model were clearly
shown in Table 5. Frontiers in Big Data 3.4 Evaluating the volatility model During the data processing, the model loss for each training and
testing data was shown in Figure 9. After adjusting the heteroscedasticity effect within the residual
ARFIMA, a comparison of ARFIMA-GARCH and ARFIMA-LSTM
models was conducted to evaluate their performance. Figure 10
graphically showed this comparison, including the actual inflation
rate data, ARFIMA(1, 0.4941, 1), ARFIMA(1, 0.4941, 1)-LSTM, and
ARFIMA(1, 0.4941, 1)-GARCH(1, 0). These four representations
were respectively depicted in blue, black, red, and purple. A model
that closely associated with the actual value and accurately tracked
its movements would likely be more accurate. Figure 9 showed the training process, which exhibited a
substantial decrease in error from 0.45 to ∼0.10 on LSTM neural
network. However, during testing, the output error proved to be
lower than those observed during training. The testing process
indicated a strong reduction in the output error from 0.42
to ∼0.05, surpassing the performance of the training process. Consequently, validation suggested that the error in testing
would be smaller compared to the training. Based on the result,
the constructed LSTM neural network effectively recognized
new data and reached the actual values through numerical
processing. The black, red, and purple lines in Figure 10 represented
ARFIMA,
ARFIMA-LSTM,
and
ARFIMA-GARCH
models,
respectively. These lines smoothly estimated the blue line (actual Frontiers in Big Data 12 frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 FIGURE 8
The ACF and PACF plot of residual data of ARFIMA model. FIGURE 8
The ACF and PACF plot of residual data of ARFIMA model. TABLE 5 Parameter settings for LSTM model. value) using a numerical model to imitate and recognize the actual
value. This indicated that the improved models represented by the
red and purple lines closely approximated the blue line, surpassing
the accuracy of the black line. Essentially, the red line performed
better than the purple line in estimating the blue line. In other
words, the sensitivity of residual data, representing volatility data,
could be effectively handled through the application of numerical
models, specifically LSTM neural network. The validity of this
statement was supported by evaluating the model using metrics
such as MSE, MAE, and MAPE. A comparison between ARFIMA
and ARFIMA-LSTM could be seen in Table 7. Frontiers in Big Data frontiersin.org 3.4 Evaluating the volatility model Based on Table 7 that tells about inflation data in Indonesia,
the United States inflation model is also built employing
ARFIMA-GARCH and ARFIMA-LSTM. Analogically, the inflation
evaluation models in the United States are shown in Table 8. Table 8
represents the evaluation model of ARIMA, ARIMA-GARCH, and
ARIMA-LSTM models of inflation data. Based on Table 8, the
ARIMA-LSTM is still having the best performance in all proposed
model of inflation data in United States However the ARIMA
the heteroscedasticity effects and also the error of the classical
model of ARFIMA for inflation data in Indonesia and ARIMA for
inflation data in United States. In addition, the fractional time series
data modeling can be applied if the series data has the long-memory
pattern data. 4 Conclusion
I
l
i
thi
d
h
d
iti it FIGURE 9
Validation error. TABLE 6 Sensitivity evaluation inflation model of Indonesia. Experiments
ARFIMA-GARCH
ARFIMA-LSTM
Mean
Variance
Standard error
Mean
Variance
Standard error
Actual data
0 0428
0 0004
0 0193
0 0428
0 0004
0 0193 FIGURE 9
V lid ti TABLE 6 Sensitivity evaluation inflation model of Indonesia. Experiments
ARFIMA-GARCH
ARFIMA-LSTM
Mean
Variance
Standard error
Mean
Variance
Standard error
Actual data
0.0428
0.0004
0.0193
0.0428
0.0004
0.0193
1. 0.0406
0.0003
0.0171
0.0427
0.0004
0.0191
2. 0.0406
0.0003
0.0171
0.0458
0.0004
0.0190
3. 0.0406
0.0003
0.0171
0.0428
0.0004
0.0189
4. 0.0406
0.0003
0.0171
0.0457
0.0004
0.0190
5. 0.0406
0.0003
0.0171
0.0438
0.0004
0.0190 the heteroscedasticity effects and also the error of the classical
model of ARFIMA for inflation data in Indonesia and ARIMA for
inflation data in United States. In addition, the fractional time series
data modeling can be applied if the series data has the long-memory
pattern data. applied to inflation data. The assessment was based on metrics
including MSE, MAE, and MAPE. Based on Table 7 that tells about inflation data in Indonesia,
the United States inflation model is also built employing
ARFIMA-GARCH and ARFIMA-LSTM. Analogically, the inflation
evaluation models in the United States are shown in Table 8. Table 8
represents the evaluation model of ARIMA, ARIMA-GARCH, and
ARIMA-LSTM models of inflation data. Based on Table 8, the
ARIMA-LSTM is still having the best performance in all proposed
model of inflation data in United States. 3.4 Evaluating the volatility model However, the ARIMA-
GARCH does not give the better performance than the classical
model of ARIMA in adjusting the error but it can do adjustment
of the hetroscedasticity effect. Frontiers in Big Data 3.4 Evaluating the volatility model Parameters
Values
Total layers
4
The number of lags
12
The number of neurons
(16, 32, 64, 128)
Learning rate
0.001
Optimization approach
Adam
Size of batch
32
Total repetition (epochs)
300
Training data
118
Testing data
44 Parameters
Values
Total layers
4
The number of lags
12
The number of neurons
(16, 32, 64, 128)
Learning rate
0.001
Optimization approach
Adam
Size of batch
32
Total repetition (epochs)
300
Training data
118
Testing data
44 From Table 7, ARFIMA-LSTM model yielded the smallest
values for all three evaluation criteria. This outcome suggested that
employing the numerical model of LSTM neural network enhanced
and refined the predicted inflation values. After adjusting the long
memory pattern of inflation data, the residuals of ARFIMA went
through further processing using LSTM to address the vanishing
gradient issue inherent in ARFIMA volatility component, often
referred to as heteroscedasticity effects. Consequently, the preferred
LSTM neural network effectively improved the heteroscedasticity
issue
of
the
classical
ARFIMA. Mitigating
the
impact
of
heteroscedasticity was achieved through either linear or nonlinear
models (Devianto et al., 2023). The application of GARCH served
as a linear model, while using the Feed Forward Neural Network
(FFNN) represented the nonlinear. Among the models, FFNN
appeared to be the most effective for addressing heteroscedasticity when compared to GARCH and hybrid GARCH-FFNN. The
results in Table 7 also showed that the neural network employing
LSTM outperformed the classical GARCH in terms of nonlinear
modeling. The same scenario is also obtained in the financial
market, where the rapid development of artificial intelligence
(LSTM) allows for a more accurate prediction of financial market
volatility than the baseline model of GARCH (Liu et al., 2022). The
results also underscored the optimal performance achieved through
the fusion of the neural network and ARFIMA, particularly when 13 Frontiers in Big Data frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 FIGURE 9
Validation error. TABLE 6 Sensitivity evaluation inflation model of Indonesia. Experiments
ARFIMA-GARCH
ARFIMA-LSTM
Mean
Variance
Standard error
Mean
Variance
Standard error
Actual data
0.0428
0.0004
0.0193
0.0428
0.0004
0.0193
1. 0.0406
0.0003
0.0171
0.0427
0.0004
0.0191
2. 0.0406
0.0003
0.0171
0.0458
0.0004
0.0190
3. 0.0406
0.0003
0.0171
0.0428
0.0004
0.0189
4. 0.0406
0.0003
0.0171
0.0457
0.0004
0.0190
5. 0.0406
0.0003
0.0171
0.0438
0.0004
0.0190
applied to inflation data. The assessment was based on metrics
including MSE, MAE, and MAPE. frontiersin.org 4 Conclusion In conclusion, this paper proposed an enhanced sensitivity
model by incorporating Long Short-Term Memory (LSTM)
neural network and Generalized Autoregressive Conditional
Heteroscedasticity (GARCH) into a long-memory model of
ARFIMA. To achieve stability, the GARCH model will reconstruct
the volatility in residual of ARFIMA using numerical processing
of Newton Raphson and the LSTM will reconstruct the volatility in Based on these two cases about the modeling of inflation data in
Indonesia and United States, the hybridization between the classical
model and LSTM method gives the best performance in adjusting 14 frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 FIGURE 10
Comparison between actual data of inflation and ARFIMA-LSTM model. FIGURE 10
Comparison between actual data of inflation and ARFIMA-LSTM model. TABLE 7 Evaluation inflation model in Indonesia. residual of ARFIMA using the adjustment of random initial weights
until the threshold error is obtained. Recognizing the limitations
of the classical long-memory ARFIMA in accurately predicting
inflation, this study underscored the necessity for ARFIMA-LSTM
and ARFIMA-GARCH models. Model
MAE
MSE
MAPE
ARFIMA
0.0063
5.8901 e-05
16.4297
ARFIMA-GARCH
0.0043
4.0726 e-05
9.7766
ARFIMA-LSTM
0.0039
3.3326 e-05
9.5185 The proposed models, ARFIMA-LSTM and ARFIMA-GARCH,
are then compared by using Mean Absolute Error (MAE), Mean
Square Error (MSE), and Mean Absolute Percentage Error (MAPE). The results show that the ARFIMA-LSTM and ARFIMA-GARCH
improve the error and also the heteroscedasticity effect of the
classical ARFIMA model. In addition, the advantages of ARFIMA-
LSTM are to achieve of the stability by learning the previous
data with the dynamical system will increase the complexity in
processing the networks, to approximate the gradient of ARFIMA
through numerical computations until a defined threshold error
was met, to retain information and patterns in residual data caused
LSTM to effectively mitigate the heteroscedasticity issue present
in ARFIMA. Despite capturing the long-pattern data inherent
in inflation, the ARFIMA model does not adequately optimize
inflation prediction. This led to the investigation of a nonlinear
solution to the gradient problem. The method included using
the LSTM, which is known for its ability to retain information
and patterns in residual data. Due to this implementation, LSTM
effectively mitigated the heteroscedasticity problem in ARFIMA. As a result, the model handled the vanishing gradient problem,
allowing the LSTM neural network to learn and bridge considerable
temporal gaps even spanning more than 1,000 discrete time steps. TABLE 8 Evaluation inflation model in United States. 4 Conclusion Model
MAE
MSE
MAPE
ARIMA
0.0015
4.4778E-06
8.3694
ARIMA-GARCH
0.0023
12.9270E-06
13.0368
ARIMA-LSTM
0.0012
3.5897E-06
6.8719 the initial weights are random, the ideal parameters do not have
the same values across experiments, necessitating validation of the
training data while developing networks. If the error of the training
data is less than the error of the testing data, the networks are
required to be stopped and the processing repeated until the error
of the training data is more than the error of the testing data to
ensure that the networks can recognize the new data using the
obtained model of ARFIMA-LSTM. the initial weights are random, the ideal parameters do not have
the same values across experiments, necessitating validation of the
training data while developing networks. If the error of the training
data is less than the error of the testing data, the networks are
required to be stopped and the processing repeated until the error
of the training data is more than the error of the testing data to
ensure that the networks can recognize the new data using the
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of iterations persist the limitations of the ARFIMA-LSTM model,
with the number of iterations increasing as the threshold error
of the networks decreases. It will require an extended period to
determine a suitable weighting parameter. Furthermore, because The datasets presented in this study can be found in
online repositories. The names of the repository/repositories and
accession number(s) can be found at: https://www.bi.go.id/id/
statistik/indikator/data-inflasi.aspx. 15 frontiersin.org Arif et al. 10.3389/fdata.2023.1282541 10.3389/fdata.2023.1282541 Funding All
claims
expressed
in
this
article
are
solely
those
of the authors and do not necessarily represent those of
their
affiliated
organizations,
or
those
of
the
publisher,
the editors and the reviewers. Any product that may be
evaluated in this article, or claim that may be made by
its
manufacturer,
is
not
guaranteed
or
endorsed
by
the
publisher. The
author(s)
declare
financial
support
was
received
for
the
research,
authorship,
and/or
publication
of
this
article. This research was supported under the scheme of
Indonesian
Collaborative
Research
with
contract
number
B/1074/UN31.LPPM/PT.01.03/2023. Conflict of interest EA: Formal analysis, Investigation, Validation, Writing—
original draft. EH: Formal analysis, Data curation, Writing—review
& editing. DD: Formal analysis, Conceptualization, Investigation,
Methodology, Validation, Writing—original draft. MY: Formal
analysis, Methodology, Software, Visualization, Writing—original
draft. DP: Data curation, Formal analysis, Methodology, Writing—
review & editing. The
authors
declare
that
the
research
was
conducted
in the absence of any commercial or financial relationships
that
could
be
construed
as
a
potential
conflict
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https://europepmc.org/articles/pmc5383912?pdf=render
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English
| null |
High-throughput high-volume nuclear imaging for preclinical in vivo compound screening§
|
EJNMMI research
| 2,017
|
cc-by
| 7,020
|
Abstract Keywords: Nuclear imaging, Automation, Image analysis, Compound screening, Biodistribution, Pharmacokinetics * Correspondence: smacholl@invicro.com; s.macholl@qmul.ac.uk
§Presented at the EANM 2016 [1]
1inviCRO Ltd, Charterhouse Square, London EC1M 6BQ, UK
3Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary
University of London, Charterhouse Square, London EC1M 6BQ, UK
Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. Macholl et al. EJNMMI Research (2017) 7:33
DOI 10.1186/s13550-017-0281-4 Open Access High-throughput high-volume nuclear
imaging for preclinical in vivo compound
screening§ Sven Macholl1,3*
, Ciara M. Finucane1,3, Jacob Hesterman2, Stephen J. Mather3, Rachel Pauplis2, Deirdre Scully2,
Jane K. Sosabowski3 and Erwan Jouannot4 Abstract Background: Preclinical single-photon emission computed tomography (SPECT)/CT imaging studies are hampered by
low throughput, hence are found typically within small volume feasibility studies. Here, imaging and image analysis
procedures are presented that allow profiling of a large volume of radiolabelled compounds within a reasonably short
total study time. Particular emphasis was put on quality control (QC) and on fast and unbiased image analysis. Methods: 2–3 His-tagged proteins were simultaneously radiolabelled by 99mTc-tricarbonyl methodology and injected
intravenously (20 nmol/kg; 100 MBq; n = 3) into patient-derived xenograft (PDX) mouse models. Whole-body SPECT/CT
images of 3 mice simultaneously were acquired 1, 4, and 24 h post-injection, extended to 48 h and/or by 0–2 h dynamic
SPECT for pre-selected compounds. Organ uptake was quantified by automated multi-atlas and manual segmentations. Data were plotted automatically, quality controlled and stored on a collaborative image management platform. Ex vivo
uptake data were collected semi-automatically and analysis performed as for imaging data. Results: >500 single animal SPECT images were acquired for 25 proteins over 5 weeks, eventually generating >3500 ROI
and >1000 items of tissue data. SPECT/CT images clearly visualized uptake in tumour and other tissues even at 48 h
post-injection. Intersubject uptake variability was typically 13% (coefficient of variation, COV). Imaging results correlated
well with ex vivo data. Conclusions: The large data set of tumour, background and systemic uptake/clearance data from 75 mice for 25
compounds allows identification of compounds of interest. The number of animals required was reduced considerably
by longitudinal imaging compared to dissection experiments. All experimental work and analyses were accomplished
within 3 months expected to be compatible with drug development programmes. QC along all workflow steps, blinding
of the imaging contract research organization to compound properties and automation provide confidence in the data
set. Additional ex vivo data were useful as a control but could be omitted from future studies in the same centre. For
even larger compound libraries, radiolabelling could be expedited and the number of imaging time points adapted to
increase weekly throughput. Multi-atlas segmentation could be expanded via SPECT/MRI; however, this would require an
MRI-compatible mouse hotel. Finally, analysis of nuclear images of radiopharmaceuticals in clinical trials may benefit from
the automated analysis procedures developed. * Correspondence: smacholl@invicro.com; s.macholl@qmul.ac.uk
§Presented at the EANM 2016 [1]
1inviCRO Ltd, Charterhouse Square, London EC1M 6BQ, UK
3Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary
University of London, Charterhouse Square, London EC1M 6BQ, UK
Full list of author information is available at the end of the article Radiolabelling
ll
l Small aliquots of all 25 proteins were radiolabelled first
for testing, following the recommendations on 99mTc-tri-
carbonyl labelling given in [19]. Subsequently, larger ali-
quots were radiolabelled for imaging. On each imaging
start day, usually 2 or 3 proteins (1.6 to 1.9 nmol of
each) were radiolabelled using 1 Isolink kit (Paul Scher-
rer Institute, Switzerland) and 2.5 GBq 99mTc-pertechne-
tate. After 2 h incubation (3 h for 5 compounds to yield
sufficient labelling efficiency), the product was purified
on a NAP-5 column (GE Healthcare). Aliquots of the
product solution were used for instant thin layer chro-
matography
(ITLC)
and
for
size
exclusion
high-
performance liquid chromatography (HPLC). The re-
mainder of typically 500 to 600 μg was split into 3 por-
tions for injections. See Additional file 1 for further
details. g
g
g
One high-throughput 18F positron emission tomography
(PET) study has been reported [18] where 42 compounds
were imaged within 11 days, at up to 3 h pi. The impres-
sive throughput did not include time for image analysis
which in that study was performed manually and thus
would prolong total study time considerably. Therefore,
besides employing longitudinal, multi-animal imaging and
efficient radiolabelling, overall study throughput was
increased in our study by automated image analysis. Macholl et al. EJNMMI Research (2017) 7:33 Macholl et al. EJNMMI Research (2017) 7:33 Page 2 of 9 Page 2 of 9 Background Furthermore, quality control was implemented at all rele-
vant steps within the whole process to ensure high data
quality. g
In vivo tissue biodistribution of administered drug or
imaging agent candidates is fundamental for their
pharmacological assessment [2], and its evaluation is
required for the translation from in vitro research to
the clinic, see e.g. [3] on radiopharmaceuticals [4]. Medicinal chemists also study compound biodistribu-
tion as an in vivo screen following in vitro tests
(which e.g. confirm target binding). The in vivo as-
sessment focusses on pharmacokinetics, here, bioavail-
ability
(concentration
in
plasma),
delivery
to
and
retention at the target and off-targets, and systemic
clearance over time. This assessment may also include
study of metabolism (DMPK). Experimental methods
can be distinguished [2, 5] e.g. between ex vivo (e.g. autoradiography [6]) and in vivo, and between label-
free (e.g. Mass Spectrometry Imaging [7]) and labelled
[8]. All these methods have their merits and draw-
backs, but generally, they are all low in throughput
[9]. That low throughput makes these tools unattract-
ive for the screening of larger combinatorial libraries
of compounds. One solution is replacement by in
silico or in vitro screening, but results of current
techniques may not be reliable enough [10]. In vivo
optical imaging may be chosen to increase throughput
[11], but this comes at the cost of limited spatial
resolution and limited quantitation [12]. In this study,
radionuclear imaging has been chosen which provides
quantitative data [13] and for which radiolabelling
automation is possible [14]. In this study, workflows, methods and software have
been developed to allow efficient high-volume compound
screening of 25 proteins by SPECT/CT of a tumour xeno-
graft mouse model. The main interest of this study was to
explore pharmacokinetics and tumour uptake in vivo in a
large panel of compounds with different biologic properties
(e.g. size, charge, and format). Specific goals were to (1)
quantify the test item uptake to the tumour in a patient-
derived xenograph (PDX) mouse model, (2) quantify sys-
temic biodistribution over up to 2 days post-injection (pi),
and (3) confirm last time point imaging data by ex vivo
analysis. The ultimate aim for the medicinal chemists and
pharmacologists was guidance for further drug design
efforts. These efforts, including the preceding, mandatory
in vitro characterization of this compound library, target
validation and animal model validation, will be described
elsewhere by Sanofi affiliated researchers. Test compounds All 25 proteins were designed and synthesized including
a hexahistidine sequence (His-tag) serving two purposes:
improving compound purification and enabling radiola-
belling by the
99mTc-tricarbonyl methodology. These
His-tagged proteins had been used in preceding in vitro
target binding affinity studies. Radiochemistry, imaging
and image analysis staff were blinded to compound
properties except for the individual molecular weights
(14 to 86 kDa). Another important consideration is the large number
of animals required for an ex vivo study to cover a suffi-
cient number of time points and to yield statistical sig-
nificance
[15]. Humane
animal
research
demands
observing the replacement, reduction and refinement
(3Rs) [16] which includes reducing the number of ani-
mals. This reduction may be achieved by non-invasive
imaging allowing repeated, longitudinal probing in each
animal post-compound administration. As a result, the
reduction factor equals the number of time points. For
example, the NCRI guidelines recommend to use 2 to 3
animals at 5 to 8 time points for a pharmacokinetic
study [17]. This corresponds to 10 to 24 animals in total
with an ex vivo method compared to 2 to 3 animals with
longitudinal in vivo imaging. Animal model Fig. 1 Examples for scheduling radiolabelling, injections and in
vivo imaging up to 5 h pi (initial part of the whole schedule). a Two compounds with initial dynamic imaging over 2 h. b Three
compounds with SPECT acquisition starting at 1 h pi All animal procedures were approved by the Animal
Welfare and Ethical Review Body at Queen Mary Uni-
versity of London and by the UK Home Office in ac-
cordance with EU Directive 2010/63/EU. Female Fox
Chase SCID mice (Charles River, UK) were 6–8 weeks
old on arrival. A first batch of 8 mice was shaved on the
lower back (1 cm2), local anaesthetic cream (lidocaine
and prilocaine) applied, and a≈10 μL fragment of pri-
mary human colon adenocarcinoma tissue (from patient
CR-IGR-034P, tumour collection CReMEC, Oncodesign,
[22]) injected subcutaneously in an intrascapular pos-
ition by trocar under isoflurane anaesthesia. The wound
was closed with spray plaster, and animals were moni-
tored more frequently for 2 days. Once tumours had
grown to 8–10 mm diameter, one animal was sacrificed
each week, the tumour excised and cut into ≈10 μL
pieces for direct passaging into a batch of 20–24 animals
(5 batches in total), following the same inoculation pro-
cedure as above. All animals underwent regular body
weight and tumour size measurements and examinations
for any signs of abnormalities. Fig. 1 Examples for scheduling radiolabelling, injections and in
vivo imaging up to 5 h pi (initial part of the whole schedule). a Two compounds with initial dynamic imaging over 2 h. b Three
compounds with SPECT acquisition starting at 1 h pi and with exact cone beam Filtered Back Projection
(VivoQuant, inviCRO LLC, USA), respectively. Reducing renal uptake To reduce renal uptake of test items, aqueous L-lysine
monohydrochloride (Sigma-Aldrich) solution (pH 7.3) Macholl et al. EJNMMI Research (2017) 7:33 Page 3 of 9 and with exact cone beam Filtered Back Projection
(VivoQuant, inviCRO LLC, USA), respectively. Fig. 1 Examples for scheduling radiolabelling, injections and in
vivo imaging up to 5 h pi (initial part of the whole schedule). a Two compounds with initial dynamic imaging over 2 h. b Three
compounds with SPECT acquisition starting at 1 h pi mixed with gelofusine (Gelaspan, B. Braun Melsungen,
Germany) was administered [20, 21] as intravenous (iv)
bolus at 4 mL/kg 30 min before radiotracer injection. Mass doses were 0.1 g/kg gelofusine and 1 g/kg L-lysine. Fig. 1 Examples for scheduling radiolabelling, injections and in
vivo imaging up to 5 h pi (initial part of the whole schedule). a Two compounds with initial dynamic imaging over 2 h. b Three
compounds with SPECT acquisition starting at 1 h pi Imaging For each compound, three mice bearing a tumour of
≈0.1 to 0.4 mL were selected randomly, weighed and
bolus iv injected with lysine/gelofusine and the test item
(5 mL/kg, 20 nmol/kg protein, ≈100 MBq) into each of
the lateral tail veins. The delivered test item doses were
calculated from the syringe weights and radioactivity
measurements before and after injections. All three mice
were imaged together on a multi-mouse bed (Minerve,
France) equipped with anaesthesia system (≈2% isoflur-
ane in 1.5 L/min medical oxygen) and warm air ventila-
tion, on a NanoSPECT/CT camera (Bioscan Inc., USA). Tissue uptake data from SPECT images were gener-
ated via multi-atlas segmentation: a reference library of
ROIs is built in usually 10 to 20 animals to create an
ROI atlas, which is co-registered to SPECT/CT images. See Additional file 1 for further details. Data of all image ROIs (volume, % ID, % ID/mL, SUV)
were saved, then custom plotted via MATLAB script,
e.g. ordered by group (i.e. compound) or by ROI class
(i.e. organ, tissue). The first SPECT image was acquired either at 1 h pi
for 50 min (radiotracer injection and first hour thereafter
without anaesthesia) or as a series of 10 SPECT images
between 10 and 110 min pi (radiotracer injected under
anaesthesia which continued for the dynamic imaging
scan), followed by a CT scan (10 min, 240 projections
with 1 s exposure to 55 kVp X-rays). Further SPECT/CT
images were acquired at 4 and 24 h pi and for some
compounds at 48 h pi (always 50 min SPECT). This
schedule allowed for staggering of 2 or 3 groups of
animals (i.e. compounds) at a time, see Fig. 1. Image analysis All image processing was performed in VivoQuant 2.0
(inviCRO) and iPACS (inviCRO). Preprocessing included
SPECT/CT coregistration at a voxel size of (0.4 mm)3
and splitting into individual mouse images. An iPACS
script facilitated entering injection doses and body
weights simultaneously for automatic conversion of
image data into absolute radioactivity, percent injected
dose (ID) or standardized uptake value (SUV). A Vivo-
Quant script generated and saved rotating maximum in-
tensity projection (MIP) movies and single-slice images
in all three orientations centred on the tumour ROI with
a chosen colour scale (here 0.2 to 20% ID/mL). γ-Counting and analysis Animals were dissected after imaging. Tissue sample
weights were transferred by push-button from the balance
into a spreadsheet template. Decay-corrected counts-per-
minute data of the daily batch of tissue samples (≈100 for 2
compounds) were measured on a γ-counter LKB Wallac
1282 Compugamma within 2 h. All accumulated data of
the whole study were then imported into MATLAB by a
custom-written script linking data points from different SPECT and CT images were reconstructed with an
iterative algorithm (HiSPECT, Scivis GmbH, Germany) Page 4 of 9 Macholl et al. EJNMMI Research (2017) 7:33 Page 4 of 9 Results Fig. 2 Exemplary images. a “Raw” SPECT/CT image of three mice as
maximum intensity projection. b CT image overlaid with 3D ROI
volumes of whole tumour, heart and kidneys, and of small liver and
muscle portions Animal model sources via compound and mouse identifiers. Uptake data
(% ID, % ID/g and SUV) were tabulated and plotted. Further details including tissue-specific calculations are
described in the Additional file 1. The preparations provided an adequate rolling stock of
animals with tumour xenografts despite the challenge of
an occasionally variable engraftment latency period
apparently not uncommon for PDX models [23]. No
complications were encountered with animal procedures
except for one ulcerating tumour (excluded from study). Median tumour growth duration was 23 days. Unfore-
seen logistical circumstances required expedition of the
study leading to seven compounds screened in the last
week. The increased demand in animals was met by
accepting some tumours outside the target size range
into the study, see abscissa of left panel in Fig. 4. These
cases are readily identifiable in the available scatter plots,
and no noticeable impact on uptake was found. Injections and imaging
Th
h
t
d
i
d Radiotracer injection quality was checked by SPECT. Both the SPECT and CT scanner units passed all manu-
facturer recommended QC procedures checked before
and after the study. Directly after image acquisition and
reconstruction, images were inspected to rule out tech-
nical issues like motion artefacts or poor injections. QC
after image analysis included (1) inspection of all MIP
movies and single slice images to confirm proper pro-
cessing (e.g. coregistration and ROI placements) and
image labelling by subject identifiers (comparing all
images per mouse) and (2) automated detection of
potential outliers in the uptake data. The phantom-derived quantification calibration factor
obtained before the in vivo study was consistent with
historic data for that scanner and with a confirmatory
calibration check after the in vivo study (within 4%). Figure 2a shows an example of a SPECT/CT image of
the three mice in the hotel before splitting into individ-
ual animals. Excellent image contrast is obtained in the
SPECT image for tumours, kidneys and bladders. Typ-
ical three-dimensional ROIs are shown in Fig. 2b on a
CT image. Eventually >500 single animal SPECT/CT im-
ages were acquired and analysed for the 25 test com-
pounds within 5 weeks. All images were made available
on the iPACS for manual inspections and archiving. They were also arranged in annotated slides decks with Ex vivo analysis included automated QC checks on γ-
counting rates falling into the linear range of the
detector and being well above background, and detec-
tion of potential outliers in the tissue weight and uptake
data. Finally, all plots were inspected visually. A
B
Fig. 2 Exemplary images. a “Raw” SPECT/CT image of three mice as
maximum intensity projection. b CT image overlaid with 3D ROI
volumes of whole tumour, heart and kidneys, and of small liver and
muscle portions A B B Statistical analysis and data plotting Basic descriptive statistical calculations were performed
in MATLAB R2015b (The MathWorks) and Excel 2013
(Microsoft). Data are reported as mean ± standard devi-
ation (SD) if not noted otherwise. Intersubject variability
was expressed as % COV (coefficient of variation) for
each group of three mice, then taking the median for
each uptake measure over all time points, compounds
and organs. Details and all organ-specific data are given
in the Additional file 1. Bland-Altman analysis and plot-
ting were conducted in Prism 5 (GraphPad Software). All other plots were prepared in MATLAB. Quality control procedures and calibrations Radiolabelling quality control (QC) included (1) ITLC
and HPLC of the product to confirm sufficient radio-
chemical purity (>95%), and (2) measurement of the
radioactivity concentration for the preparation of indi-
vidual radioactivity doses (target≈100 MBq) at a volume
dose of ≈0.2 mL. Mice were monitored daily to control health status,
and mice were enrolled into the study only in absence of
any abnormalities. QC of tumour volume estimates by
calliper measurements was by SPECT/CT and weighing
excised tumours (target range 0.1 to 0.4 mL). Radiolabelling The preparations for imaging had the following study
averages: radiochemical purity = (98.0 ± 1.6) %, activity
= (331 ± 82) MBq in (539 ± 59) μL for three injections. Macholl et al. EJNMMI Research (2017) 7:33 Page 5 of 9 Page 5 of 9 Page 5 of 9 rotating MIP and single-slice image views as SPECT,
SPECT/CT and ROI/CT. rotating MIP and single-slice image views as SPECT,
SPECT/CT and ROI/CT. high activity samples to sufficiently reduce γ-counter
dead-time. Data of such repeat measurements automat-
ically replaced QC-rejected data of the first counting
run. Image analysis produced SPECT data of ≈4000 ROIs. Examples for SPECT data plots are provided in Fig. 3a,
b. While each plot condenses information from at least
9 SPECT images (three animals, three time points) or
even from ≈500 (all animals, all time points as in Fig. 3a),
the subdivision into tissues and compounds, and the
choice of parameters and various plot types resulted
again in an expansion, here to >1000 plots. This was
managed by tables of contents in PDF slide decks with a
hierarchical folder-structure allowing quick access to any
plot of interest. The final data set was visualized in an automatically
generated PDF slide deck of 172 plots, see examples in
Fig. 3c, d. Biodistribution data from ex vivo dissection and
SPECT were compared by Bland-Altman analysis, see
Fig. 4 for examples. Discussion
Study results Intersubject variability for tumour, heart, kidneys and
liver was 13% for all considered uptake measures (% ID,
% ID/mL, and SUV), ranging from 7% for liver SUV to
19% for heart SUV. Preliminary test radiolabelling proved useful in cases
where insufficient radiolabelling efficiency was solved by
longer incubation. Subsequent radiopreparations for im-
aging all passed QC. The only problem encountered with the PDX mouse
model was engraftment latency in some animals of the
first passage. Despite an initially reduced choice of ani-
mals for imaging, significant study delays were avoided. In parallel to regular animal welfare checks, an efficient
and frequent stock checking procedure allowed to Ex vivo analysis Left panel, tumour weight (ex vivo) and tumour volume (SPECT ROI) (converted to weight assuming tissue density = 1.0 g/mL); right panel,
radiotracer uptake in tumour as % ID (ex vivo from gamma counting and in vivo from SPECT ROI). Bias as dotted line (at −80 mg and +0.14% ID, respectively),
95% agreement interval as pair of bold dotted lines. Values on the abscissa are individual tumour averages over the two measurement methods SPECT signal distribution. Therefore, a fixed volume
encompassing ROI was used. continuously adapt the schedule for tumour inoculations
and for optimal selection of animals for imaging. This study did not attempt to study the effect of lysine
and gelofusine on the biodistribution but rather utilized
this as a blanket method to reduce kidney uptake. Con-
trol experiments to quantitate the absolute or relative
kidney uptake reduction with different radiotracers, e.g. compounds of different molecular weight, were out of
scope because they would have doubled the number of
experiments. For the presented study, the kidney uptake
data have to be interpreted very carefully. However, since
renal clearance is downstream of delivery to other tis-
sues of interest (e.g. tumour), no significant effect is ex-
pected on these. For subcutaneous tumours with their often inhomo-
geneous SPECT signal distribution, inconsistent size and
shape and more variable location, ROIs were generated
manually. Still, for the voxel size and tumour sizes in
this study, erosion of or dilation by a single voxel layer
from the surface of a 3D ROI changes volume estimates
by tens of percents. Therefore, ROI volume estimates
were compared to ex vivo tumour weights as QC. In
cases of large discrepancy, the tumour ROI was revisited
and, if necessary, edited. Multi-atlas segmentation has proven useful for auto-
mated region identification especially in clinical neuro-
applications [24–27]. To overcome the limitation of
many available software packages designed for clinical
brain imaging, this approach has been implemented and
employed for full and subregion segmentation of other
organs (for whole body distribution and radiation dosim-
etry) in several non-human species. Data variability—due
to factors such as animal positioning, the non-rigidity of
non-brain regions, general shape/distribution outliers
and
fluctuating
contrast-to-noise—may
occasionally
cause segmentation failures, thus requiring strict QC
and manual corrections. Ex vivo analysis Circa 1000 tissue samples were collected, weighed and
counted, in typical daily batches of ≈100 or 150 samples. All data analysis was automated and results were avail-
able in near real-time. However, often a delay of 1–2 days
between sampling and counting was required for very Tumor
Muscle
Liver
Kidneys
Heart
Exvivo
Imaging
B
A
D
C
Fig. 3 Exemplary plots of imaging data (top row, mean and SEM error bars for N = 3) and ex vivo data (bottom row, individual data points). a % ID/mL in
left kidney ROIs (all compounds, all time points). b % ID/mL data of compound 17 (all ROIs, all time points). c % ID/g in liver in all animals (≈24 or 48 h pi). d % ID/g in tumour in all animals (≈24 or 48 h pi) Imaging
A Tumor
Muscle
Liver
Kidneys
Heart
B A B Exvivo
C D D C Fig. 3 Exemplary plots of imaging data (top row, mean and SEM error bars for N = 3) and ex vivo data (bottom row, individual data points). a % ID/mL in
left kidney ROIs (all compounds, all time points). b % ID/mL data of compound 17 (all ROIs, all time points). c % ID/g in liver in all animals (≈24 or 48 h pi). d % ID/g in tumour in all animals (≈24 or 48 h pi) Macholl et al. EJNMMI Research (2017) 7:33 Page 6 of 9 Fig. 4 Bland-Altman plots on the difference in tumour data between ex vivo (dissected tissue) and in vivo (SPECT/CT) methods, based on pairs of data from
the same animal. Left panel, tumour weight (ex vivo) and tumour volume (SPECT ROI) (converted to weight assuming tissue density = 1.0 g/mL); right panel,
radiotracer uptake in tumour as % ID (ex vivo from gamma counting and in vivo from SPECT ROI). Bias as dotted line (at −80 mg and +0.14% ID, respectively),
95% agreement interval as pair of bold dotted lines. Values on the abscissa are individual tumour averages over the two measurement methods Fig. 4 Bland-Altman plots on the difference in tumour data between ex vivo (dissected tissue) and in vivo (SPECT/CT) methods, based on pairs of data from
the same animal. Ex vivo analysis Nevertheless, we found in tim-
ing experiments (not shown here) that multi-atlas seg-
mentation followed by user editing of the automatically
generated ROIs still significantly decreases processing
time and observer variability compared to segmenting
regions manually de novo. The initial study design was based on 13 SPECT im-
ages (of the mouse hotel, N = 3 mice) per compound
starting with dynamic scanning over the first 2 h post-
radiotracer injection and ending at 48 h pi. To cut
costs and to reduce stress to animals, this imaging
schedule was reduced in the final study plan for 18 of
the 25 compounds by removing the last time point
and/or reducing the initial dynamic scanning to a 1 h
“static” image acquisition. Detailed comparisons of
compounds with these 2 different first image acquisi-
tions may need to consider the different durations
under isoflurane anaesthesia. Conclusions Feasibility and application of high-volume in vivo com-
pound screening by preclinical SPECT/CT and auto-
mated analysis have been demonstrated. Excluding the
animal model setup, the required time for such a study
including a final report is on the order of weeks. Import-
antly, longitudinal imaging reduced the number of ani-
mals three- to fourfold compared to dissection studies,
even 12- to 13-fold when considering the dynamic scans
in this study. Specific for this study was the compound type, large
proteins, for which (a) a test on intact biological func-
tionality after radiolabelling may be skipped (or replaced
by in vitro target binding affinity measurements of the
His-tagged but unlabelled compounds as done here),
and (b) a “one size fits all” radiolabelling procedure may
work. Subsequent use of the data Compound ranking, or identifying compounds fulfilling
a set of inclusion criteria, or elimination of poorly per-
forming compounds can be based on algorithms com-
bining several uptake, uptake rate or clearance measures
and limits. A separate paper will apply such an algorithm
in combination with in vitro and in silico measures. Follow-up in vivo experiments related to pharmacokinet-
ics may be performed on a selected small group of po-
tential lead candidates, e.g. on compound/radiolabel
stability in vivo. Ex vivo Ex vivo data were acquired to validate the biodistribution
results from imaging which was successful as Bland-
Altman analysis showed. One observation was, however,
that the SPECT tumour ROI volume tended to be signifi-
cantly larger than the ex vivo tumour weight. This discrep-
ancy is likely due to an overestimation of the tumour
volume on the images, and deviation from the assumed
1 g/mL mass density may also play a role. Such an overes-
timated ROI volume likely captures spillover of the
tumour SPECT signal just outside the tumour, an occa-
sional artefact from image reconstruction or smoothing. This conjecture would explain the good match of tumour
uptake values (% ID) between ex vivo and in vivo measure-
ments and also explain the observed non-equivalent mea-
sures of uptake concentration in tumour (ex vivo % ID/g
versus imaging % ID/mL). As discussed above, the ROI
drawing protocol can be tailored to generate ROIs that
achieve a better match in either % ID (as for tumours in
this study) or % ID/g values between ex vivo dissection
and in vivo imaging data. Image analysis Region of interest definition methods were chosen to
strike a balance between precision, consistency and effi-
ciency. Concentration (% ID/g, SUV) estimates allow the
use of fixed volume regions. These benefit from gener-
ally low anatomical intersubject variability in preclinical
studies. Examples are liver and kidneys. For liver with its
complex organ shape, a subregional ROI near the organ
centre was used under the assumption of homogeneous
distribution throughout the organ tissue. Kidneys have a
simpler organ shape but can exhibit inhomogeneous Optimal subject number is a challenging question for
preclinical studies. A larger number of mice as normally
used in a dissection study allows for better inference
towards the mouse population compared to the often
reduced number of mice used for imaging. On the one
hand, an animal group size N > 3 is suggested in some
circumstances [15, 28]. On the other hand, longitudinal
imaging of a single mouse removes that intersubject
variability and may allow for a clearer observation e.g. of Macholl et al. EJNMMI Research (2017) 7:33 Page 7 of 9 Page 7 of 9 radiotracer kinetics [29]. In this study, statistical analysis
was utilized but not an overarching driver. Rather, the
goal was to achieve sufficient consistency and power in
methodology to identify compounds of interest to be
evaluated more thoroughly in follow-up experiments
and with additional in vitro results at hand. Figure 3a, c
shows that indeed relevant, apparent differences between
compounds are often much greater than intersubject
variability at the chosen group size of three. Ultimately,
the choice of the group size represents a compromise
between statistical needs for in vivo and ex vivo study
design and study aim, logistical implications, cost and
ethics. imaging needs to decay considerably to reach the measure-
ment window of a γ-counter. This slows down throughput,
but after successful validation as in this study, ex vivo ana-
lysis could be omitted altogether from future imaging
studies. High volume imaging generates large amounts of data,
but for preclinical SPECT/CT or PET/CT, this is well
manageable within a contemporary standard information
technology environment. References 1. Macholl S, Finucane CM, Mather SJ, Hesterman J, Scully D, Jouannot E. From
compound library to lead compound selection via established 99mTc
radiochemistry, high-throughput preclinical SPECT/CT imaging and
automated analysis. Eur J Nucl Med Mol Imaging. 2016;43:S221. http://dx. doi.org/10.1007/s00259-016-3484-4. 2. Lanao JM, Fraile MA. Drug tissue distribution: study methods and
therapeutic implications. Curr Pharm Des. 2005;11:3829–45. 2. Lanao JM, Fraile MA. Drug tissue distribution: study methods and
therapeutic implications. Curr Pharm Des. 2005;11:3829–45. 3. Todde S, Windhorst AD, Behe M, Bormans G, Decristoforo C, Faivre-Chauvet
A, et al. EANM guideline for the preparation of an investigational medicinal
product dossier (IMPD). Eur J Nucl Med Mol Imaging. 2014;41:2175–85. 4. Reichel A, Lienau P. Pharmacokinetics in drug discovery: an exposure-
centred approach to optimising and predicting drug efficacy and safety. In:
Nielsch U, Fuhrmann U, Jaroch S, editors. New approaches drug discov. Cham: Springer International Publishing; 2015. p. 235–60. Available from:
http://link.springer.com/10.1007/164_2015_26. (cited 2016 Nov 6). 2. Lanao JM, Fraile MA. Drug tissue distribution: study methods and
therapeutic implications. Curr Pharm Des. 2005;11:3829–45. 3. Todde S, Windhorst AD, Behe M, Bormans G, Decristoforo C, Faivre-Chauvet
A, et al. EANM guideline for the preparation of an investigational medicinal
product dossier (IMPD). Eur J Nucl Med Mol Imaging. 2014;41:2175–85. 4. Reichel A, Lienau P. Pharmacokinetics in drug discovery: an exposure-
centred approach to optimising and predicting drug efficacy and safety. In:
Nielsch U, Fuhrmann U, Jaroch S, editors. New approaches drug discov. Cham: Springer International Publishing; 2015. p. 235–60. Available from:
http://link.springer.com/10.1007/164_2015_26. (cited 2016 Nov 6). Ethics approval Ethics approval
All applicable international, national and/or institutional guidelines for the
care and use of animals were followed. Acknowledgements We are indebted to Dr. Emmanuelle Vigne (Sanofi-Aventis) for providing the
compounds and to Dr. Jane Sosabowski (QMUL) for making the excellent
preclinical imaging facility at QMUL available for contract research. Many
thanks to Dr. Roxana Kashani (QMUL) for radiochemistry support, to Ed
Waters (now KCL) for technical support and to Dr. Julie Foster and the
Animal Technician Service team (QMUL) for biology support. 9. Alavijeh MS, Palmer AM. The pivotal role of drug metabolism and
pharmacokinetics in the discovery and development of new medicines. IDrugs Investig Drugs J. 2004;7:755–63. Throughput All experiments from animal model setup to final ana-
lysis took 3 months. This included time for the animal
model setup which could be eliminated in other studies
when using naïve animals, or which might take longer
for certain disease models. The whole process proved efficient and robust. High
confidence in the data set was achieved by (1) blinding
the contract researchers to compound properties, (2)
quality control checks at all stages, (3) transparency of
raw data and analysis procedures and easy access to all
data, and (4) a high automation level in data acquisition
and analysis. This study design for large proteins included imaging
up to 48 h pi. In hindsight, this time point adds only
limited information to that obtained at 24 h and could
be omitted in a future study of such compounds, making
space in the schedule available for other experiments. Additional ex vivo data validated the image analysis
approach. While systematic differences in the output pa-
rameters can be observed, these can be corrected for if
desired, and they are not expected to affect the relative
compound ranking. Future studies employing this im-
aging workflow may be streamlined by removing such ex
vivo analysis, although other complementary ex vivo Image analysis of incoming data was done within a day
to create updated study plots. A fortunate benefit was
that image analysis was performed in a later time zone
than data acquisition (GMT→EST), effectively stretch-
ing the working day. γ-counters are more sensitive than SPECT cameras. Therefore, the fairly large injected radioactivity dose for Page 8 of 9 Page 8 of 9 Page 8 of 9 Macholl et al. EJNMMI Research (2017) 7:33 Macholl et al. EJNMMI Research (2017) 7:33 Page 8 of 9 techniques like high-resolution autoradiography could
be added. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Additional file Additional file 1: Detailed descriptions of the radiolabelling procedure,
image analysis, γ-counting procedure and analysis, and intersubject
variability estimation. Further results of the γ-counter calibration, ex
vivo/imaging comparison and intersubject variability. (DOCX 171 kb) Additional file 1: Detailed descriptions of the radiolabelling procedure,
image analysis, γ-counting procedure and analysis, and intersubject
variability estimation. Further results of the γ-counter calibration, ex
vivo/imaging comparison and intersubject variability. (DOCX 171 kb) p
(
)
g g
;
4. Reichel A, Lienau P. Pharmacokinetics in drug discovery: an exposure-
centred approach to optimising and predicting drug efficacy and safety. In:
Nielsch U, Fuhrmann U, Jaroch S, editors. New approaches drug discov. Cham: Springer International Publishing; 2015. p. 235–60. Available from:
http://link.springer.com/10.1007/164_2015_26. (cited 2016 Nov 6). 4. Reichel A, Lienau P. Pharmacokinetics in drug discovery: an exposure-
centred approach to optimising and predicting drug efficacy and safety. In:
Nielsch U, Fuhrmann U, Jaroch S, editors. New approaches drug discov. Cham: Springer International Publishing; 2015. p. 235–60. Available from:
http://link.springer.com/10.1007/164_2015_26. (cited 2016 Nov 6). Competing interests g
SJM and JKS contributed to this work as consultants to inviCRO. All other
authors are full-time employees at the industrial research organizations de-
tailed in the affiliations section. Abbreviations
l 5. Joseph SB, Prabhu S, Boswell CA. Biodistribution of therapeutic biologics:
methods and applications in informing target biology, pharmacokinetics,
and dosing strategies. In: Gad SC, editor. Pharm. Sci. Encycl. Hoboken: Wiley;
2015. p. 1–14. Available from: http://doi.wiley.com/10.1002/9780470571224. pse542. (cited 2016 Nov 10). 3Rs: Replacement, reduction and refinement; COV: Coefficient of variation;
CT: Computed tomography; HPLC: High-performance liquid chromatography;
ID: Injected dose; ITLC: Instant thin layer chromatography; iv: Intravenous;
MIP: Maximum intensity projection; MRI: Magnetic resonance imaging;
PACS: Picture Archiving and Communication System; PDX: Patient-derived
xenograft; QC: Quality control; ROI: Region of interest; SCID: Severe
combined immunodeficiency; SD: Standard deviation; SPECT: Single-photon
emission computed tomography; SUV: Standardized uptake value 6. Solon EG. Use of radioactive compounds and autoradiography to determine
drug tissue distribution. Chem Res Toxicol. 2012;25:543–55. 7. Nilsson A, Goodwin RJA, Shariatgorji M, Vallianatou T, Webborn PJH,
Andrén PE. Mass spectrometry imaging in drug development. Anal
Chem. 2015;87:1437–55. combined immunodeficiency; SD: Standard deviation; SPECT: Single-photon
emission computed tomography; SUV: Standardized uptake value 8. Isin EM, Elmore CS, Nilsson GN, Thompson RA, Weidolf L. Use of
radiolabeled compounds in drug metabolism and pharmacokinetic studies. Chem Res Toxicol. 2012;25:532–42. 8. Isin EM, Elmore CS, Nilsson GN, Thompson RA, Weidolf L. Use of
radiolabeled compounds in drug metabolism and pharmacokinetic studies. Chem Res Toxicol. 2012;25:532–42. Author details
1 1inviCRO Ltd, Charterhouse Square, London EC1M 6BQ, UK. 2inviCRO, LLC, 27
Dry Dock Avenue, 7th Floor West, Boston, MA 02210, USA. 3Centre for
Molecular Oncology, Barts Cancer Institute, Queen Mary University of
London, Charterhouse Square, London EC1M 6BQ, UK. 4Sanofi Aventis
Recherche Développement, 1, Avenue Pierre Brossolette, 91380
Chilly-Mazarin, France. Received: 27 February 2017 Accepted: 22 March 2017 Received: 27 February 2017 Accepted: 22 March 2017 Consent for publication
Not applicable. Consent for publication
Not applicable. Image acquisition throughput could be increased further
by adapting and/or reducing the number of imaging
time points (as done in this study shortening 0…2 h
dynamic to 1–2 h static scans) and by increasing the
number of animals in the field of view (e.g. with a 3D-
printed 4-bed hotel (inviCRO)). Additional automated
analyses can be integrated into the workflow at little cost
in extra time, for example kinetic modelling (with
image-derived input function) or radiation dosimetry. Analysis of dual isotope images can be done practically
as quickly as that of single isotope images, but the study
schedule would have to factor in additional radiochemis-
try resources and development time. Additional valuable
information might be extracted from SPECT images
when replacing CT by anatomical MR images with su-
perior soft tissue contrast, ideally in future with a mouse
hotel compatible MRI setup and with simultaneous MR
and nuclear image acquisitions. Finally, analysis of
nuclear images of radiopharmaceuticals in clinical trials
may benefit from the automated analysis procedures
developed. Authors’ contributions EJ, CMF and SJM designed the research. SJM and JKS conducted the
radiochemistry work. CMF and SM performed the in vivo and ex vivo
experiments. JH and DS wrote software. JH, RP and DS analysed the images. JH and SM analysed the ex vivo data. SM performed statistical analyses. SM
and JH wrote the paper. All authors read and approved the final manuscript. 13. Szanda I. Quantification in nuclear preclinical imaging. Handb. Small Anim. Imaging. CRC Press; 2016. p. 409–22. Available from: http://dx.doi.org/10. 1201/b19052-30. (cited 2016 Nov 12). Page 9 of 9 Macholl et al. EJNMMI Research (2017) 7:33 14. Macholl S, Glaser M. Radiochemistry for preclinical imaging studies. Handb. Small Anim. Imaging. CRC Press; 2016. p. 277–314. Available from: http://dx. doi.org/10.1201/b19052-22. (cited 2016 Apr 15). . Eckelman WC, Kilbourn MR, Joyal JL, Labiris R, Valliant JF. Justifying 15. Eckelman WC, Kilbourn MR, Joyal JL, Labiris R, Valliant JF. Justifying the
number of animals for each experiment. Nucl Med Biol. 2007;34:229–32. 15. Eckelman WC, Kilbourn MR, Joyal JL, Labiris R, Valliant JF. Justifying the
number of animals for each experiment. Nucl Med Biol. 2007;34:229–32. number of animals for each experiment. Nucl Med Biol. 2007;34:229– 16. Hendee WR. Ethics and regulations for research with animals. Handb. Small
Anim. Imaging. CRC Press; 2016. p. 7–16. Available from: http://dx.doi.org/
10.1201/b19052-4. (cited 2016 Apr 15). 17. Workman P, Aboagye EO, Balkwill F, Balmain A, Bruder G, Chaplin DJ, et al. Guidelines for the welfare and use of animals in cancer research. Br J
Cancer. 2010;102:1555–77. 18. Gagnon MKJ, Hausner SH, Marik J, Abbey CK, Marshall JF, Sutcliffe JL. High-
throughput in vivo screening of targeted molecular imaging agents. Proc
Natl Acad Sci. 2009;106:17904–9. 19. Badar A, Williams J, de Rosales RT, Tavaré R, Kampmeier F, Blower PJ, et al. Optimising the radiolabelling properties of technetium tricarbonyl and His-
tagged proteins. EJNMMI Res. 2014;4:14. 20. Melis M, Bijster M, de Visser M, Konijnenberg MW, de Swart J, Rolleman EJ,
et al. Dose–response effect of Gelofusine on renal uptake and retention of
radiolabelled octreotate in rats with CA20948 tumours. Eur J Nucl Med Mol
Imaging. 2009;36:1968–76. 21. Rolleman EJ, Bernard BF, Breeman WAP, Forrer F, de Blois E, Hoppin J, et al. Molecular imaging of reduced renal uptake of radiolabelled
[DOTA0,Tyr3]octreotate by the combination of lysine and Gelofusine in rats. Nukl Nucl Med. 2008;47:110–5. 22. Macholl et al. EJNMMI Research (2017) 7:33 Authors’ contributions Julien S, Merino-Trigo A, Lacroix L, Pocard M, Goere D, Mariani P, et al. Characterization of a large panel of patient-derived tumor xenografts
representing the clinical heterogeneity of human colorectal cancer. Clin
Cancer Res. 2012;18:5314–28. 22. Julien S, Merino-Trigo A, Lacroix L, Pocard M, Goere D, Mariani P, et al. Characterization of a large panel of patient-derived tumor xenografts
representing the clinical heterogeneity of human colorectal cancer. Clin
Cancer Res. 2012;18:5314–28. 23. Siolas D, Hannon GJ. Patient-derived tumor xenografts: transforming clinical
samples into mouse models. Cancer Res. 2013;73:5315–9. 23. Siolas D, Hannon GJ. Patient-derived tumor xenografts: transforming clinica
samples into mouse models. Cancer Res. 2013;73:5315–9. 24. Aljabar P, Heckemann RA, Hammers A, Hajnal JV, Rueckert D. Multi-atlas
based segmentation of brain images: atlas selection and its effect on
accuracy. NeuroImage. 2009;46:726–38. 25. Artaechevarria X, Munoz-Barrutia A, Ortiz-de-Solorzano C. Combination
strategies in multi-atlas image segmentation: application to brain MR data. IEEE Trans Med Imaging. 2009;28:1266–77. 26. Heckemann RA, Hajnal JV, Aljabar P, Rueckert D, Hammers A. Automatic
anatomical brain MRI segmentation combining label propagation and
decision fusion. NeuroImage. 2006;33:115–26. 27. Rohlfing T, Brandt R, Menzel R, Maurer CR. Evaluation of atlas selection
strategies for atlas-based image segmentation with application to confocal
microscopy images of bee brains. NeuroImage. 2004;21:1428–42. 28. Soares EJ, Hesterman J, Hoppin J. The use of power analysis in small sample
pre-clinical imaging studies. Mol Imaging Biol. 2016;18:448. 28. Soares EJ, Hesterman J, Hoppin J. The use of power analysis in small sample
pre-clinical imaging studies. Mol Imaging Biol. 2016;18:448. 29. Scheibe PO. Number of samples—hypothesis testing. Nucl Med Biol. 2008;35:3–9. 29. Scheibe PO. Number of samples—hypothesis testing. Nucl Med Biol. 2008;35:3–9. Submit your manuscript to a
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TARPTAUTINĖ SOCIALINIO TYRIMO PROGRAMA IR GALIMYBĖS POLITOLOGINEI ANALIZEI
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TARPTAUTINĖ SOCIALINIO TYRIMO
PROGRAMA IR GALIMYBĖS POLITOLOGINEI
ANALIZEI1 Tarptautinė socialinio tyrimo programa (angl. The International
Social Survey Programme, ISSP) yra tęstinė kasmetinė tarptautinė
apklausų bendradarbiavimo programa, apimanti svarbiausias socia
linių tyrimų temas. Ji sieja esamus socialinių mokslų projektus ir
koordinuoja būsimų tyrimų tikslus, taip papildydama individualius
nacionalinius tyrimus tarptautine ir tarpkultūrine perspektyva. (Online) ISSN 2424-6034 (Online) ISSN 2424-6034 (Online) ISSN 2424-6034 1
Informacija parengta Kauno technologijos universiteto Viešosios politikos ir admi
nistravimo institutui vykdant projektą „Tarptautinė socialinio tyrimo programa: pi
lietiškumo, darbo ir socialinės gerovės vertinimai Lietuvoje (ISSP LT-CIWO)“, kurį
finansuoja Lietuvos mokslo taryba (sutartis Nr. MIP-082/2014). DOI: https://doi.org/10.15388/Polit.2017.1.10676
1
Informacija parengta Kauno technologijos universiteto Viešosios politikos ir admi
nistravimo institutui vykdant projektą „Tarptautinė socialinio tyrimo programa: pi
lietiškumo, darbo ir socialinės gerovės vertinimai Lietuvoje (ISSP LT-CIWO)“, kurį
finansuoja Lietuvos mokslo taryba (sutartis Nr. MIP-082/2014). DOI: https://doi.org/10.15388/Polit.2017.1.10676 2
Smith T. W., „The ISSP: History, Organisation and Members, Working Principles and
Outcomes. A Historical-sociological Account“, Haller M., Jowell R., Smith T. W.
(eds.), The International Social Survey Programme 1984–2009: Charting the Globe,
London: Routledge, 2012. 3
www.issp.org 4
www.issp.org/page.php?pageId=241 Tarptautinės socialinio tyrimo programos
istorija ir metodologija Tarptautinės socialinio tyrimo programos (TSTP) pradžia yra 1984 m.,
kai susirinkę sociologai ir politologai iš Australijos, Didžiosios Bri
tanijos, JAV bei Vokietijos sutarė, kad reikėtų kasmet įgyvendinti
tam tikrai tematikai skirtą tarptautinį modulį, leidžiantį atlikti tarp
tautinius palyginamuosius tyrimus. Nutarta, kad toks tyrimas turė
tų tapti jau vykdomų nacionalinių socialinių apklausų (Australijos
nacionalinio socialinių mokslų tyrimo, Britų socialinių nuostatų ty 202 P O L I TO LO G I J A 2017/1 ( 85 ) rimo bei JAV ir Vokietijoje atliekamo Bendrojo socialinio tyrimo)
sudedamąja dalimi2. Pirmajam TSTP tyrimui 1985 m. buvo pasi
rinktas klausimų modulis „Valdžios vaidmuo“, be jau keturių šalių
steigėjų, dar apklausti Austrijos ir Italijos gyventojai. Praėjus trims
dešimtmečiams ši programa vienija mokslininkus, atliekančius kas
metinius tarptautinius palyginamuosius tyrimus beveik 50 šalių3. Sprendimai dėl TSTP įgyvendinimo žingsnių yra priimami kas
met vykstančioje Generalinėje asamblėjoje. Generalinėje asamblė
joje atstovaujama kiekvienai šaliai, esančiai TSTP nare. Kiekviena
šalis turi teisę siųsti ne daugiau kaip trijų žmonių delegaciją, tačiau
šaliai yra sutiekiamas tik vienas balsas. Lietuvai TSTP Generalinėje
asamblėjoje atstovauja prof. dr. Algis Krupavičius (Vytauto Didžio
jo universitetas) ir doc. dr. Eglė Butkevičienė (Kauno technologijos
universitetas)4. Generalinės asamblėjos metu yra svarstomas ir prii
mamas kitų metų tyrimo modulis, kurį parengia asamblėjoje sudaryta
darbo grupė, vyksta Metodologinio ir kitų komitetų posėdžiai. Asam
blėjos metu taip pat diskutuojama TSTP metodologiniais klausimais,
gautų tyrimo rezultatų temomis. 2016 m. organizuoti TSTP Gene
ralinę asamblėją buvo patikėta Lietuvai, tai byloja apie mūsų šalies
tyrėjų TSTP komandos pripažinimą patikimu partneriu. 2016 m. ba
landžio 30–gegužės 4 d. Kaune vykusioje Generalinėje asamblėjoje,
surengtoje bendradarbiaujant KTU Socialinių, humanitarinių mokslų
ir menų bei VDU Socialinių mokslų fakultetams, lankėsi daugiau nei
70 dalyvių iš 40 valstybių. TSTP pripažįstama kaip viena iš pagrindinių pasaulinių socia
linių problemų stebėsenos tyrimų sistemų, o jos tyrimų išskirtinu 203 RECENZIJOS IR APŽVALGOS mą lemia tarptautinis duomenų palyginamumas. Įgyvendinant šią
programą, yra taikomi dideli metodologiniai tarptautinių apklausų
atlikimo reikalavimai. Šiam tyrimui yra būdingas ne tik platus su
tam tikra socialine problematika susijusių temų spektras, bet ir kar
totinumas bėgant laikui, nes kas 5–7 metai tas pats TSTP modulis
paprastai yra pakartojamas. Kartotinio modulio statusas suteikia
mas tada, kai 2/3 jo klausimų yra paimti iš anksčiau naudotų vieno
ar kelių tos pačios tematikos klausimų modulių. Nuo 1985 m. iki 2015 m. jau yra atliktos daugiau nei trys de
šimtys kasmetinių apklausų. 5
www.gesis.org/en/issp Tarptautinės socialinio tyrimo programos
istorija ir metodologija Skirtingose pasaulio šalyse keturis
kartus buvo įgyvendintas modulis „Socialinė nelygybė“, „Šeima ir
kintantys lyčių vaidmenys“, „Valdžios vaidmuo“ ir „Darbo orien
tacijos“; tris kartus – „Religija“, „Aplinka“ ir „Nacionalinė tapa
tybė“; du kartus – „Pilietiškumas“ ir „Socialiniai tinklai“, po kar
tą – „Laisvalaikis ir sportas“ bei „Sveikata ir sveikatos priežiūra“. 2016 m. Tarptautinės socialinio tyrimo programos apklausos tema
buvo penktą kartą atliekamas pirmojo modulio „Valdžios vaidmuo“
tyrimas. Beje, Generalinėje asamblėjoje sudarytoje darbo grupė
je, kuri buvo atsakinga už šio modulio klausimyno parengimą, be
Švedijos, Didžiosios Britanijos, Prancūzijos, Meksikos, Ispanijos ir
Japonijos mokslininkų, Lietuvai atstovavo prof. dr. A. Krupavičius
bei doc. dr. E. Butkevičienė. Tai dar vienas Lietuvos tyrėjų pripaži
nimo tarptautiniu mastu indikatorius. Visose šalyse atliktų apklausų duomenys yra deponuojami į
Duomenų archyvą – Leibnico socialinių mokslų institutą Vokie
tijoje5. Archyve iš visų šalių perduoti duomenys yra patikrinami,
parengiami sujungti duomenų failai, jie aprašomi ir kartu su klau
simynais nacionalinėmis kalbomis, informacija apie tyrimo dizai
ną ir kita su tyrimų atlikimu susijusia dokumentacija publikuojami
antrinei duomenų analizei atlikti. Visi TSTP apklausų duomenys 204 P O L I TO LO G I J A 2017/1 ( 85 ) yra vieši ir naudojami geriausios kokybės istorinei ir tarptautinei
palyginamajai socialinių problemų analizei atlikti bei mokslinėms
publikacijoms, kurių skelbta daugiau nei 7 000, parengti6. 6
Žr. The 2016 ISSP Bibliography: A Report (www.issp.org/uploads/editor_uploads/
files/ISSPBB16rpt.doc).
7
2010–2013 m. programos įgyvendinimas finansuotas laimėjus Lietuvos mokslo tary
bos (LMT) projektus: 2010–2011 m. „Socialinių problemų stebėsena: Tarptautinės
socialinio tyrimo programos įgyvendinimas (SPS)“, 2012–2013 m. – „Tarptautinė
socialinio tyrimo programa: Lietuvos socialinių problemų stebėsena (ISSP-LT)“.
Nuo 2014 m. Tarptautinė socialinio tyrimo programa įgyvendinama vykdant projek
tą „Tarptautinė socialinio tyrimo programa: pilietiškumo, darbo ir socialinės gerovės
vertinimai Lietuvoje“, kurį taip pat finansuoja LMT. 6
Žr. The 2016 ISSP Bibliography: A Report (www.issp.org/uploads/editor_uploads/
files/ISSPBB16rpt.doc).i Tarptautinės socialinio tyrimo programos įgyvendinimas
Lietuvoje Lietuva į Tarptautinę socialinio tyrimo programą įsitraukė ne visai
sklandžiai. 1994 m. Lietuvoje buvo atlikta viena apklausa, tačiau jos
duomenų Vokietijos archyve nėra. Oficialiai Lietuva į TSTP įsitrau
kė 2010 m., kai KTU Politikos ir viešojo administravimo institutas
(po 2014 m. KTU įvykusių struktūrinių pertvarkymų – Viešosios
politikos ir administravimo institutas) tapo Tarptautinės socialinio
tyrimo programos nariu ir Lietuvoje pradėjo įgyvendinti kasmeti
nes apklausas7. 2010–2016 m. yra įgyvendinti 8 moduliai. Pirmai
siais narystės metais Lietuvoje atliktas vadinamasis „besivejantis“
tyrimas, t. y. 2010 m. įgyvendintas ir tais metais numatytas tyrimų
modulis „Aplinka“, ir „Socialinės nelygybės“ modulis, kurį TSTP
dalyvaujančios šalys atliko 2009 m. 2011 m. įgyvendintas modulis
„Sveikata ir sveikatos priežiūra“, 2012 m. – „Šeima, darbas ir lyčių
vaidmenys“, 2013 m. – „Nacionalinė tapatybė“. 2014 m. atlikta Lie
tuvos gyventojų apklausa tema „Pilietiškumas“ ir 2015 m. „Darbo
orientacijos“, o 2016 m. – „Valdžios vaidmuo“. Toliau straipsnyje bus trumpai pristatytos trys politikos mokslų 205 RECENZIJOS IR APŽVALGOS bendruomenei svarbios ir įdomios apklausos temomis „Nacionalinė
tapatybė“, „Pilietiškumas“ bei „Valdžios vaidmuo“. 2013 m. Tarptautinės socialinio tyrimo programos
modulis „Nacionalinė tapatybė“ Tyrimo „Nacionalinė tapatybė“ tikslas yra ištirti Lietuvos gyventojų
santykį su Lietuva, jų požiūrį į kitas valstybes ir tarptautines orga
nizacijas bei į Lietuvoje gyvenančias ne lietuvių tautybės žmonių
grupes. Pirmiausia tirtas požiūris į lietuvybę ir į savo šalį. Respondentų
klausta, kiek jie jaučiasi artimi savo gyvenvietei, miestui, savo savi
valdybei, savo valstybei ir Europai. Lietuvos gyventojai vertino, kiek
svarbu yra gimti Lietuvoje, turėti Lietuvos pilietybę, gyventi Lietu
voje didžiąją gyvenimo dalį, mokėti kalbėti lietuviškai, būti kataliku
ir kt. Toliau teirautasi, kiek respondentai didžiuojasi Lietuva dėl jos
politinės įtakos pasaulyje, ekonominių pasiekimų, socialinės apsau
gos sistemos ir dėl to, kaip joje veikia demokratija. Toliau klausta,
kiek didžiuojamasi valstybe dėl savo šalies mokslinių ir technologi
nių pasiekimų, karinių pajėgų, šalies sporto bei meno ir literatūros,
istorijos pasiekimų. Atskiras dėmesys buvo skirtas Lietuvos santykiams su kitomis
valstybėmis ir tarptautinėmis organizacijomis. Domėtasi, ar valstybė
turėtų riboti užsienietiškų produktų importą tam, kad apsaugotų savo
nacionalinę ekonomiką. Klausta, ar tarptautinės organizacijos turėtų
turėti teisę priversti vykdyti sprendimus, susijusius su kai kuriomis
problemomis, pavyzdžiui, aplinkos tarša. Apklausos metu buvo pa
liestas ir draudimo parduoti žemę užsieniečiams klausimas. Vertinta,
ar didelės tarptautinės įmonės daro vis daugiau žalos vietiniam Lietu
vos verslui. Teirautasi, ar šalis turėtų laikytis sprendimų tų tarptautinių
organizacijų, kurioms ji priklauso, net jei vyriausybė jiems nepritaria. 206 P O L I TO LO G I J A 2017/1 ( 85 ) Kitas TSTP modulio „Nacionalinė tapatybė“ blokas buvo skir
tas įvertinti Lietuvos gyventojų požiūrius į mažumas bei imigrantus. Teirautasi, ar tautinėms mažumoms turėtų būti teikiama vyriausybės
parama jų papročiams ir tradicijoms išsaugoti. Prašyta atsakyti, ar
šaliai yra geriau, kai skirtingos rasinės ir tautinės grupės išlaiko sa
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nių. Klausta, ar imigrantai praturtina visuomenę, atsinešdami naujas
idėjas ir kultūrą, ar, atvirkščiai, ją griauna. Paskutinė tema skirta požiūriui į ES tirti. Aiškintasi, ar buvimas
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modulis „Pilietiškumas“ Tyrimo „Pilietiškumas“ tikslas yra ištirti Lietuvos gyventojų sociali
nį ir politinį aktyvumą bei požiūrį į piliečio ir valstybės santykį. Apklausa pradėta klausiant, kiek svarbu yra visada balsuoti rin
kimuose, niekada nebandyti išsisukti nuo mokesčių, visada paklus
ti šalies įstatymams ir teisės aktams. Toliau respondentai vertino
nuolatinio valdžios veiksmų stebėjimo, aktyvaus dalyvavimo so
cialinėse ar politinėse asociacijose ir bandymo suprasti kitaip mąs
tančių žmonių argumentus reikšmingumą ir kt. Pateikus skirtingų
politinio ir socialinio veikimo formų sąrašą (pavyzdžiui, peticijos
pasirašymas, dalyvavimas demonstracijoje ar politiniame susirin
kime ar mitinge ir kt.), respondentų teirautasi, ar jie darė kurį nors
iš nurodytų veiksmų. Dar viena tyrimo tema – priklausomybė skir
tingų tipų grupėms ar asociacijoms ir aktyvumas jose: politinėse 207 RECENZIJOS IR APŽVALGOS partijose, profesinėse sąjungose, verslo ar profesinėse asociacijose,
Bažnyčioje ar kitose religinėse organizacijose, sporto, laisvalaikio
ar kultūrinėse grupėse. Kitas klausimų blokas buvo skirtas ištirti žmogaus teisių reikš
mingumą demokratijoje. Pirmiausia klausta, kiek yra svarbu, kad
visi piliečiai turėtų tinkamą pragyvenimo lygį ir kad valdžia gerbtų
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įsitraukti į pilietinio nepaklusnumo veiklas. Tirta, kiek yra svarbu,
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kaip jie nuomonė apie tai, ką daro valdžia, nieko nereiškia, ir ar val
džiai rūpi, ką galvoja tokie žmonės kaip patys respondentai. Vertinta,
ar apklausti Lietuvos gyventojai pakankamai gerai supranta svarbius
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modulis „Valdžios vaidmuo“ Tyrimo „Valdžios vaidmuo“ tikslas yra ištirti, kaip Lietuvos gyven
tojai vertina valdžią, už kokių funkcijų įgyvendinimą valdžia turėtų 208 P O L I TO LO G I J A 2017/1 ( 85 ) būti atsakinga ir išsiaiškinti žmonių požiūrį į pilietines laisves ir vi
suomenės saugumą. Pirmiausia teirautasi nuomonės, ar gali būti leidžiama organizuoti
viešus susirinkimus ar protesto eitynes ir demonstracijas protestuo
jant prieš valdžią, jei žmonės griežtai prieštarauja valdžios veiks
mams. Atskiras klausimų blokas skirtas valdžios galimybėms imtis
įvairių veiksmų ekonomikos srityje (pavyzdžiui, valdžios finansavi
mas projektams naujoms darbo vietoms sukurti, parama nuosmukį
patiriančioms pramonės šakoms, siekiant išsaugoti darbo vietas, ir
kt.). Aiškintasi, ar respondentai norėtų didesnių ar mažesnių išlaidų
įvairiose valdžios išlaidų srityse. Tirtos valdžios atsakomybės ribos,
pavyzdžiui, suteikiant darbą kiekvienam norinčiam dirbti, mažinant
pajamų skirtumus tarp turtingų ir vargšų, suteikiant finansinę paramą
universitetų studentams iš mažas pajamas turinčių šeimų, skatinant
vyrų ir moterų lygybę ir kt. Norėta sužinoti, koks turėtų būti val
džios vaidmuo teikiant sveikatos priežiūrą sergantiesiems, vyresnio
amžiaus žmonių priežiūrą bei vaikų mokyklinį ugdymą. Pateikus
sąrašą žmonių ir organizacijų (pavyzdžiui, žiniasklaida, profesinės
sąjungos, verslas, bankai ir pramonė ir kt.), prašyta pasakyti, kas turi
didžiausią ir antrą didžiausią įtaką Lietuvos valdžios veiksmams. Tiriant nuomonę apie pilietines laisves ir visuomenės saugumą
pirmiausia paklausta, ar Lietuvos valdžia turėtų viešose vietose ste
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laiškus ir kitą informaciją, kuria keičiamasi internete. Aiškintasi,
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nalinio saugumo valdžia turėtų turėti teisę rinkti informaciją apie bet
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aktas. Neliko pamiršta ir klasikinių klausimų grupė apie politiką. Tei
rautasi, kiek respondentai asmeniškai domisi politika. Vertinta as
meninė politinė kompetencija ir galima įtaka valdžios sprendimams. 209 RECENZIJOS IR APŽVALGOS Toliau aiškintasi dabartinių mokesčių Lietuvoje teisingumas dideles,
vidutines ir mažas pajamas gaunantiems žmonėms. Tirtas požiūris
apie didžiųjų privačių įmonių Lietuvoje elgesį, t. y. kaip dažnai jos
laikosi įstatymų ir kitų teisės aktų, taip pat ar mėgina vengti mo
kėti mokesčius. Užduoti keli klausimai ir apie Lietuvos politikų bei
valdžios pareigūnų korupciją. Apklausos pabaigoje aiškintasi, kaip
sėkmingai Lietuvos valdžia veikia suteikdama sveikatos priežiūros
paslaugas sergantiems bei tinkamą pragyvenimo lygį senyvo am
žiaus žmonėms ir šalindama grėsmes Lietuvos saugumui. Prieiga prie Tarptautinės socialinio tyrimo programos
duomenų Lietuvos socialinių mokslų akademinė bendruomenė savišvietos,
mokymo bei mokslo tikslais gali naudotis reprezentatyvių Lietuvos
gyventojų apklausų duomenimis lietuvių kalba, kurie prieinami Hu
manitarinių ir socialinių mokslų duomenų archyvo (LiDA) apklausų
duomenų kataloge (www.lidata.eu/QUANT). Visi apklausų duome
nys tarptautinei palyginamajai analizei atlikti yra prieinami TSTP
Duomenų archyvo kataloge Vokietijoje (zacat.gesis.org). GIEDRIUS ŽVALIAUSKAS
Kauno technologijos universitetas
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The cascade methods of doubly-fed induction machine for generator system
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International Journal of Power Electronics and Drive Systems/International Journal of Electrical and Computer Engineering
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International Journal of Power Electronics and Drive System (IJPEDS)
Vol. 12, No. 1, Mar 2021, pp. 112~120
ISSN: 2088-8694, DOI: 10.11591/ijpeds.v12.i1.pp112-120 International Journal of Power Electronics and Drive System (IJPEDS)
Vol. 12, No. 1, Mar 2021, pp. 112~120
ISSN: 2088-8694, DOI: 10.11591/ijpeds.v12.i1.pp112-120 International Journal of Power Electronics and Drive System (IJPEDS)
Vol. 12, No. 1, Mar 2021, pp. 112~120
ISSN: 2088-8694, DOI: 10.11591/ijpeds.v12.i1.pp112-120 112 112 ABSTRACT This paper presents two solutions to cascade the doubly-fed induction
machines in the power generator systems. The first solution is a traditional
one with the power control circuit located on the stator-side. The second
solution is a new one with the power control circuit located on the rotor-side. After analysis and evaluation, it is shown that the solution with the power
control circuit located on the rotor side has advantages over the solution with
the power control circuit located on the stator-side. Therefore, the authors
chose the solution which is a power control circuit located on the rotor side
to study, analyze in-depth and run the simulation. The results show that the
proposed solution has a very good quality, the output voltage of the generator
always follows the grid-voltage even when changing the gird-voltage or
changing the speed of the generator. Keywords:
Active power
BDFIG
DFIG
Excitation control
Reactive power
Similar signals This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author:
Diep-Dung Nguyen
Faculty of Electrical-Electronic Engineering
Vietnam Maritime University, Vietnam
Email: nguyendiepdung.smq@gmail.com Diep-Dung Nguyen
Faculty of Electrical-Electronic Engineering
Vietnam Maritime University, Vietnam
Email: nguyendiepdung.smq@gmail.com The cascade methods of doubly-fed induction machine for
generator system Diep-Dung Nguyen1, Ngoc-Hoan Than2, Duc-Tuan Hoang3
1,3Faculty of Electrical-Electronic Engineering, Vietnam Maritime University, Vietnam
2Departement of Electrical Engineering and Automation, Haiphong Private University, Vietnam Diep-Dung Nguyen1, Ngoc-Hoan Than2, Duc-Tuan Hoang3
1,3Faculty of Electrical-Electronic Engineering, Vietnam Maritime University, Vietnam
2Departement of Electrical Engineering and Automation, Haiphong Private University, Vietnam Journal homepage: http://ijpeds.iaescore.com 1.
INTRODUCTION However, the technique of controlling DFIG rotor circuits is very difficult [16], especially when the
rotor speed changes, the controller must control the frequency of the rotor circuit changes fast and timely in
order to synchronize the generator voltage with the grid voltage. Figure 1. The power generator system using DFIG Figure 1. The power generator system using DFIG A new, effective solution has been proposed which is to use the cascade of two DFIGs. The
outstanding advantage of DFIG cascade solution is that the system has the ability to stabilize the frequency of
the generator voltage very well when the rotor speed changes. There are two solutions to cascade DFIGs: the
first solution is a traditional one with the power control circuit located on the stator-side, the second solution
is a new one with the power control circuit located on the rotor-side. This research will analyze the
characteristics of two DFIG cascade solutions, then choose the appropriate solution to improve the efficiency
of the generator system, study in-depth and simulation the chosen solution. 1.
INTRODUCTION Nowadays, the resources of fuel are running out, so it is very important and necessary to find and
use the renewable sources. These renewable energy sources must be able to connect to the grid or work
parallel together [1], [2]. When changing the rotor speed or unstable grid voltage, it is very difficult to control
the generator system in order for the generator-voltage coincide with the grid-voltage [3], [4]. The method
that uses the doubly-fed induction machine (DFIM) in generator mode is a very effective [5]-[7], because it
can keep the stable frequency of the generator-voltage when changing the rotor speed [8]-[10]. p
q
y
g
g
g g
p
The DFIM is an induction machine with both stator and rotor wire [11], [12]. The DFIM worked in
the generator mode (DFIG) has inherent advantages, such as the ability to keep the unchanged frequency of
the voltage in the case of variable rotor speed, a small control circuit. Therefore, DFIG is widely used in
generator systems, such as the wind generator, shaft generator in a ship. In the generator system using DFIG,
the power control circuit is located in the rotor-side, and the generated power in the stator-side transmits
directly to the grid. Thus, the power of the control circuit is much lower than the power fed into the grid. Some research [13]-[15] have succeeded in controlling DFIG as a generator. The diagram of a power
generator system using DFIG is shown in Figure 1. Stator of DFIG is directly connected to the grid. The rotor of DFIG is connected to the grid via a
power control circuit that let the energy can transmit in both directions. There are two working modes of
DFIG: the over synchronization mode and the below synchronization mode. In the both modes, the stator
generates the energy fed into the grid. The rotor receives the energy in the over synchronous mode and
transmits the energy in the below synchronous mode. DFIG as a generator has been increasingly applied in Journal homepage: http://ijpeds.iaescore.com
113 113 ISSN: 2088-8694 Int J Pow Elec & Dri Syst practice. However, the technique of controlling DFIG rotor circuits is very difficult [16], especially when the
rotor speed changes, the controller must control the frequency of the rotor circuit changes fast and timely in
order to synchronize the generator voltage with the grid voltage. practice. The cascade methods of doubly-fed induction machine for generator system (Diep-Dung Nguyen) 2.1. The structure of control system 2.1. The structure of control system The system is described in Figure 2 [17]. The system consists of two DFIGs. Two rotor-shafts are
tightly connected together and the electric-wire of two rotors are also directly connected to each other. The
power control circuit is located on the stator-side of DFIG1. The electrical power generated in the stator of
DFIG 2 is transmitted directly to the grid. The natural feature of the DFIG cascade system is that the synchronization between the stator
voltage of DFIG2 and grid voltage is very high and stable [18]. Furthermore, the quality of DFIG cascade is
much better than the generator system using the independent DFIG [19]. Therefore, this system is being
applied more and more widely in grid-connected generators. Today, several manufacturing facilities have
integrated the DFIG cascade system into the one machine which is called Brushless Doubly-Fed Induction
Generator (BDFIG), the diagram of BDFIG is shown in Figure 3 [17]. In Figure 3, the stator of DFIG1 has the number of pole pairs p1=2, the stator of DFIG2 has the
number of pole pairs p2=1. The electric-wire in the rotor of two DFIGs are directly connected together, so
this system hasn't a ring and brush. Therefore, this system is called Brushless Doubly-Fed Induction
Generator (BDFIG). The BDFIG fabrication techniques are presented in detail in the research [20]. The
number of pole pairs of DFIG1 and DFIG2 can be equal or different and there are two ways to connect two
electric-wire of rotors, so there are four solutions of integration BDFIG, which are listed in Table 1 [21], [22] The cascade methods of doubly-fed induction machine for generator system (Diep-Dung Nguyen)
114 114 ISSN: 2088-8694 Figure 2. The DFIG cascade system with the power
control circuit located on the stator-side
Figure 3. The structure of BDFIG Figure 2. The DFIG cascade system with the power
control circuit located on the stator-side Figure 3. The structure of BDFIG Table 1. The Types of BDFIG
The connection between two rotors
p1= p2
The sync speed
Ps1 (pu)
Ps2 (pu)
Pg=Ps1 +Ps2
Similar-phase
Yes
No production
g
p
g
p
−
0
Similar-phase
No
p
g
−
g
p
1
g
p
2
−
g
p
. 2.1. The structure of control system
Reverse phase
Yes
p
g
2
g
p
−
g
p
−
g
p
. 2
−
Reverse phase
No
p
g
g
p
1
−
g
p
2
−
g
p
.
− where p1, p2 are the number of polar pairs of DFIG1 and DFIG2 respectively; Ps1, Ps2 and Pg are the active
power of DFIG1-stator, DFIG2-stator, and the grid respectively; 𝜔 is the angular speed of the rotor; 𝜔𝑔 is the
angular speed of the grid-voltage. where p1, p2 are the number of polar pairs of DFIG1 and DFIG2 respectively; Ps1, Ps2 and Pg are the active
power of DFIG1-stator, DFIG2-stator, and the grid respectively; 𝜔 is the angular speed of the rotor; 𝜔𝑔 is the
angular speed of the grid-voltage. g
p
g
g
The operation principle of BDFIG is shown in Figure 4 [17]. The stator current frequency of DFIG1
and DFIG2 are fs1, fs2 respectively; the stator current angular speed of DFIG1 and DFIG2 are 𝜔𝑠1, 𝜔𝑠2
respectively; the rotor current angular speed of DFIG1 and DFIG2 are 𝜔𝑟1, 𝜔𝑟2 respectively. Figure 4. The principle of operation of BDFIG Figure 4. The principle of operation of BDFIG Figure 4. The principle of operation of BDFIG 2.2. The energies in the system Considering a BDFIG type with the number of pole pairs is different, connecting method in the rotor
is reverse-phase. Since DFIG2 is directly connected to the grid, the angular speed of the stator circuit is equal
to the angular speed of the grid voltage 𝜔𝑠2 = 𝜔𝑔. The angular speed of the stator circuit of DFIG1 is: The angular speed of the stator circuit of DFIG1 is: 1
1
2
2
(
). s
s
p
p
=
+
−
(1) (1) 1
1
2
2
(
). s
s
p
p
=
+
− Int J Pow Elec & Dri Syst, Vol. 12, No. 1, March 2021 : 112 – 120 115 ISSN: 2088-8694 Int J Pow Elec & Dri Syst The angular speed of the rotor circuit of DFIG1 and DFIG2 are: The angular speed of the rotor circuit of DFIG1 and DFIG2 are: lar speed of the rotor circuit of DFIG1 and DFIG2 are: 2
2
2. r
s
p
=
−
(2)
1
1
2
. r
s
p
=
−
(3)
The stator power of DFIG1 and DFIG2 are: 2
2
2. r
s
p
=
− 1
1
2
. r
s
p
=
− The stator power of DFIG1 and DFIG2 are: The stator power of DFIG1 and DFIG2 are: The stator power of DFIG1 and DFIG2 are: 1
1
. . s
g
g
p
P
P
−
=
2
2
. . s
g
g
p
P
P
−
= 1
1
. . s
g
g
p
P
P
−
=
2
2
. . s
g
g
p
P
P
−
= The rotor power of DFIG1 and DFIG2 are: The rotor power of DFIG1 and DFIG2 are: 1
1
1
1
r
r
s
s
P
P
= −
(6)
2
2
2
2
r
r
s
s
P
P
= −
(7) 1
1
1
1
r
r
s
s
P
P
= −
2
2
2
2
r
r
s
s
P
P
= − 1
1
1
1
r
r
s
s
P
P
= − (6) (7) (7) The rotor of DFIG1 and DFIG2 are connected into the loop, so Pr1+Pr2=0 or Pr1=-Pr2. The
generated power of BDFIG into the grid is Pg: 1
2
g
s
s
P
P
P
=
+
(8) 1
2
g
s
s
P
P
P
=
+
(8) (8) 1
2
g
s
s
P
P
P
=
+ From (4)-(7), we have the relationship equation between the power stator of DFIG1 and DFIG2 are
as follows: 1
1
2
2
s
s
s
s
P
P
= −
(9) 1
1
2
2
s
s
s
s
P
P
= − (9) As shown in (9) also shows the relationship between the power of the control circuit and the power
transmitted into the grid. In fact, there is also a power loss in the stator and rotor of DFIG1 and DFIG2, the
energy flow diagram is shown in Figure 5 [17], [18]. (a)
(b)
Figure 5. The energy flow diagram in BDFIG; a) super_synchronous, b) sub_synchronous (a) (b) Figure 5. The energy flow diagram in BDFIG; a) super_synchronous, b) sub_synchronous According to the energy diagram, DFIG2 always generates active power, DFIG1 generates active
power in the over synchronous mode and absorbs the active power in the below synchronous mode. When we
connect the rotor wire by reverse phase method, the energy flowed through DFIG1 stator in the opposite
direction: DFIG1 absorbs active power in the over synchronous mode and generates active power in the
below synchronous mode. The cascade methods of doubly-fed induction machine for generator system (Diep-Dung Nguyen)
116 116 ISSN: 2088-8694 The research [23] has compared the quality of the generated electric between BDFIG and DFIG. The results showed that the power generator system using BDFIG has higher quality than a system using
DFIG. However, the drawback of the BDFIG system is that its large size and greater power loss compare
with DFIG. The rotor power of DFIG1 and DFIG2 are: To overcome this limitation, another solution to cascade DFIGs is proposed that is the solution
with a power control circuit located on the rotor side. This solution has reduced size, little power loss, simple
control system, etc. 3.1. The structure and operating principle 3.1. The structure and operating principle The structure of DFIG cascade with the power control circuit located on the stator-side is shown in
Figure 6. The system includes two DFIG1 and DFIG2 with the same number of pole pairs p1=p2=p, the
processing signal stages and the current control circuit. The stator of DFIG1 is connected directly to the grid
so the angular speed of the stator circuit of DFIG1 (𝜔𝑠1) is equal to the angular speed of grid voltage:
𝜔𝑠1 = 𝜔𝑔. 𝑔
The angular speed of the rotor circuit of DFIG1 𝜔𝑟1 = 𝜔𝑠1 −𝑝. 𝜔. This angular speed is kept
constant through the signal processing stage and the current control circuit, so the angular speed of the rotor
circuit of DFIG2 is 𝜔𝑟2 = 𝜔𝑟1 = (𝜔𝑠1 −𝑝. 𝜔). The angular speed of the stator circuit of DFIG2 is 𝜔𝑠2 = 𝑝. 𝜔+ 𝜔𝑟2 = 𝑝. 𝜔+ (𝜔𝑠1 −𝑝. 𝜔=
𝜔𝑠1 = 𝜔𝑔. Therefore, the angular speed of the stator circuit of DFIG2 (𝜔𝑠2) is always equal to the angular
speed of the grid voltage (𝜔𝑔), and is independent to the angular speed of rotor (𝜔). With this nature
properties, the system has the ability to follow the grid voltage very well and sustainably. In the system
structure, DFIG1 does not have the role of generating power fed into the grid but only creating the signals in
the rotor which is the input of the signal processing stage. Therefore, DFIG1 can be selected as a small size
and capacity DFIG to reduce the power loss, cost and size of the system. Figure 6. The structure of DFIG cascade with the power control circuit located on the stator-side Figure 6. The structure of DFIG cascade with the power control circuit located on the stator-side 3.2. The control system Int J Pow Elec & Dri Syst, Vol. 12, No. 1, March 2021 : 112 – 120
3.2. The control system
The detailed block diagram of the control system is shown in Figure 7 [24]. The system includes:
•
DFIG1 is a small Doubly-Fed Induction Generator, its stator is connected directly to the grid, its rotor
works in the high resistance mode. The role of DFIG1 is to create the similar signal voltage in the rotor. •
The similar an isolation stage is a signal amplification circuit with the resistance input is high in order
for the rotor of DFIG1 works in the high resistance mode. •
The integral stage
•
The signal amplification stages Kp, Kq and the invert stage (-1). •
The rotation stage of 90 degrees
•
The current control circuit creates the current fed into the rotor of DFIG2. •
DFIG2 is a Doubly-Fed Induction Generator with the role of generating the current fed into the grid
The rotor-shaft of DFIG1 and DFIG2 are rigidly connected together in order for the angular
coordinates of the rotor wire and stator wire are equal. The detailed block diagram of the control system is shown in Figure 7 [24]. The system includes:
•
DFIG1 is a small Doubly-Fed Induction Generator, its stator is connected directly to the grid, its rotor
works in the high resistance mode. The role of DFIG1 is to create the similar signal voltage in the rotor. g
g
g
•
The similar an isolation stage is a signal amplification circuit with the resistance input is high in order
for the rotor of DFIG1 works in the high resistance mode. •
The similar an isolation stage is a signal amplification circuit with the resistance input is high in order
for the rotor of DFIG1 works in the high resistance mode. •
The signal amplification stages Kp, Kq and the invert stage (-1). •
The current control circuit creates the current fed into the rotor of DFIG2. •
DFIG2 is a Doubly-Fed Induction Generator with the role of generating the current fed into the grid
The rotor-shaft of DFIG1 and DFIG2 are rigidly connected together in order for the angular
coordinates of the rotor wire and stator wire are equal. 3.3. Controlling the system With the case of the stator of DFIG2 is not connected to the grid, the phase and frequency of the
stator voltage of DFIG2 are always equal to the ones of grid voltage. The voltage amplitude of DFIG2 stator
depends on the coefficient (Kcl) in the similar an isolation stage [24]. Therefore, the voltage amplitude of
DFIG2 stator can be adjusted by adjusting the coefficient Kcl. After adjustment, Generator voltage has the
phase, amplitude and frequency equal to the ones of the grid voltage. This is a good conditional for
connecting the generator system to the grid. g
g
y
g
With the case of the stator of DFIG2 is connected to the grid, the active power and the reactive
power of DFIG2 (Ps2, Qs2) are proportional to the amplification coefficients KP, Kq [24] respectively: 2
2
. . s
p
s
q
P
K
X
Q
K Y
=
=
2
2
. . s
p
s
q
P
K
X
Q
K Y
=
=
(10.a,b) 2
2
. . s
p
s
q
P
K
X
Q
K Y
=
=
(10.a,b) (10.a,b) where X, Y is constant values and do not depend on the rotor speed, but only on the structure of DFIG1 and
DFIG2. Therefore, the active power and reactive power of DFIG2 (Ps2, Qs2) transmitted to the grid can be
independently controlled by adjusting the coefficients Kp, Kq. where X, Y is constant values and do not depend on the rotor speed, but only on the structure of DFIG1 and
DFIG2. Therefore, the active power and reactive power of DFIG2 (Ps2, Qs2) transmitted to the grid can be
independently controlled by adjusting the coefficients Kp, Kq. q
The research [25], [26] has analyzed the mathematical model of this system, then pointed out the
advantages of applying this system in the generator connected to the grid. To show more detail the operating
principle and the characteristics of the signals at all stages, we will build and simulate the system model on
Matlab Simulink. DFIG1 and DFIG2 are Asynchronous Machine in the Sim-Power-System library with the
following parameters in Table 2: Table 2. The parameters of DFIG1 and DFIG2
P(VA)
U(V)
f(HZ)
Rs (Ω)
Ls (H)
Rr(Ω)
Lr(H)
Lm(H)
DFIG1
200
600
60
0.0198
6.7e-4
0.0189
6.9e-4
0.0339
DFIG2
100000
600
60
0.0160
5.8e-4
0.0168
6.7e-4
0.0419 3.2. The control system •
DFIG2 is a Doubly-Fed Induction Generator with the role of generating the current fed into the •
DFIG2 is a Doubly-Fed Induction Generator with the role of generating the current fed into the grid
The rotor-shaft of DFIG1 and DFIG2 are rigidly connected together in order for the angular
coordinates of the rotor wire and stator wire are equal. Int J Pow Elec & Dri Syst, Vol. 12, No. 1, March 2021 : 112 – 120
117 117 Int J Pow Elec & Dri Syst ISSN: 2088-8694 Figure 7. The control system with the power control circuit located on the stator-side Figure 7. The control system with the power control circuit located on the stator-side 4.
RESULTS AND DISCUSSION The response of the system: (a) when changing the rotor speed; (b) when changing the grid voltage (a) (b) (b) Figure 9. The response of the system: (a) when changing the rotor speed; (b) when changing the grid voltage 4.
RESULTS AND DISCUSSION 4.1. In the case of the generator system is not connected to the grid The process of adjusting Kcl is shown in Figure 8. The voltage of A-phase in the stator of DFIG2
(us2a) has the frequency and phase always coincides with the ones of A-phase voltage (ug) of the grid. The
amplitude of us2a can be controlled by adjusting the coefficient Kcl. At time t = 0.7 s, setting Kcl = 11.4, then
the voltage us2a is equal to the grid voltage in amplitude, phase, and frequency. This is a good condition to
connect the generator system to the grid. The cascade methods of doubly-fed induction machine for generator system (Diep-Dung Nguye ISSN: 2088-8694 118 Figure 8. The process of adjusting Kcl Figure 8. The process of adjusting Kcl When changing the rotor speed, the response of the system is shown in Figure 9(a). The results
show that the voltage of A-phase in the stator of the generator (us2a) always coincides with the A-phase
voltage of the grid (ug) in amplitude, frequency and phase. Therefore, the ability to follow the grid voltage of
the generator DFIG2 is very good when the rotor speed changes. When changing the rotor speed, the response of the system is shown in Figure 9(a). The results
show that the voltage of A-phase in the stator of the generator (us2a) always coincides with the A-phase
voltage of the grid (ug) in amplitude, frequency and phase. Therefore, the ability to follow the grid voltage of
the generator DFIG2 is very good when the rotor speed changes. When the grid voltage drops, the response of the system is shown in Figure 9(b). The results show
that the voltage of A-phase in the stator of the generator (us2a) always coincides with the A-phase voltage of
the grid (ug) in amplitude, frequency and phase. Therefore, the ability to follow the grid voltage of the
generator DFIG2 is very good when the grid voltage changes. Therefore, in the case of stator DFIG2 is not
connected to the grid, after adjustment Kcl, the voltage of the generator system always follows the grid
voltage even when the rotor speed changes or the grid voltage changes. This is a good condition to connect
the generator system to the grid. (a)
(b)
Figure 9. 4.2. In the case of the generator system is connected to the grid With the case of generator system is connected to the grid, the response of the system is shown in
Figure 10. The results show that it is possible to independently control the active and reactive power of
DFIG2 transmitted to the grid by adjusting the coefficients Kp and Kq. The active power depends only to the
coefficient Kp. The reactive power depends only to the coefficient Kq. Therefore, controlling the active and
reactive power of DFIG2 transmitted to the grid will be very simple and convenient. From the above results,
it shows that the DFIG cascade system with the power control circuit located on the rotor-side is very suitable
for application in the generator with variable rotor speed. Int J Pow Elec & Dri Syst, Vol. 12, No. 1, March 2021 : 112 – 120 ISSN: 2088-8694 119 Int J Pow Elec & Dri Syst Figure 10. The response of the system when Kp and Kq change Figure 10. The response of the system when Kp and Kq change 5.
CONCLUSION The paper presented and analyzed the grid-connected generator structures using DFIG cascade
solutions, then shown the advantages of applying DFIG cascade solutions in generator system, especially the
cascade solution with the power control circuit located in the rotor. The paper presented and analyzed the grid-connected generator structures using DFIG cascade
solutions, then shown the advantages of applying DFIG cascade solutions in generator system, especially the
cascade solution with the power control circuit located in the rotor. p
The solution of cascade DFIG with the power control circuit at the rotor side has the following
outstanding advantages: The ability to follow the grid voltage is very good, even when the grid voltage
changes or the rotor speed changes; The active power and reactive power of the generator fed into the grid
are independently controlled through coefficients KP and Kq; The simple system control system structure. In
the future, the authors will study more in-depth the solution with the power control circuit located on the
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https://openalex.org/W2154691164
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https://bmcmedresmethodol.biomedcentral.com/counter/pdf/10.1186/s12874-015-0017-y
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English
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An application of propensity score weighting to quantify the causal effect of rectal sexually transmitted infections on incident HIV among men who have sex with men
|
BMC Medical research methodology
| 2,015
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cc-by
| 7,161
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* Correspondence: asvaugh@emory.edu
1Department of Epidemiology, Rollins School of Public Health, Emory
University, 1518 Clifton Rd NE, Atlanta 30322, GA, USA
Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access An application of propensity score weighting to
quantify the causal effect of rectal sexually
transmitted infections on incident HIV among
men who have sex with men Adam S Vaughan1*, Colleen F Kelley1,2, Nicole Luisi1, Carlos del Rio2,3, Patrick S Sullivan1 and Eli S Rosenberg1 Vaughan et al. BMC Medical Research Methodology (2015) 15:25
DOI 10.1186/s12874-015-0017-y Vaughan et al. BMC Medical Research Methodology (2015) 15:25
DOI 10.1186/s12874-015-0017-y © 2015 Vaughan et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Data sources and definitions The InvolveMENt study is a recently concluded, longitu-
dinal cohort of black and white, sexually active MSM
aged 18–39 years in Atlanta, Georgia, recruited from
community-based venues and social media between June
2010 through October 2012 [15]. MSM were tested for
HIV at enrollment using rapid antibody tests, with con-
firmatory serum CD4 and viral load measures for prelimin-
ary positives. MSM who were HIV-negative at enrollment
were included in the longitudinal portion of the study, at-
tending study visits at 3 and 6 months, and subsequent 6
month intervals for two years, or until HIV seroconversion. At the same visits, participants were tested for urethral and
rectal Neisseria gonorrhoeae (gonorrhea) and Chlamydia
trachomatis (chlamydia) using nucleic acid amplification
testing and Treponema pallidum (syphilis). using the
rapid plasma regain (RPR) test with confirmatory quanti-
tative nontreponemal titers and treponemal IgG [16,17]. At each study visit, participants completed a computer-
administered questionnaire that collected aggregate sexual
behaviors, such as the number of UAI partners, and in-
cluded a dyadic inventory of the most recent 5 sex partners
in the previous 6 months [15]. Demographic and sexual be-
haviors (i.e.: condom use, receptive and insertive sex roles)
were collected for each of these partners. Consequently, in the absence of RCT data, establishing a
causal association between a non-preventive exposure and
incident HIV requires application of epidemiologic analysis
methods that can control for time-varying behavioral con-
founding and accommodate small numbers of observed
events. The ideal data would include longitudinal evalu-
ation of the exposure, outcome, and all possible time-
varying confounders, with measurements made using
methods to minimize misclassification. Given such a data-
set, propensity score methods would address these issues
and, with confirmation of model assumptions, provide an
estimate of the causal effect of interest [8]. For this analysis, the outcome was incident HIV infec-
tion. The exposure was defined as the first (i.e. earliest)
diagnosis of incident rectal STI (either gonorrhea or
chlamydia). An STI diagnosis was considered to be inci-
dent if the individual tested negative for the same STI in
the prior interval, or if the STI diagnosis followed an ini-
tial visit with the same STI diagnosis with confirmation
of study-provided treatment. As we could not determine
the timing of the rectal STI for men who were diagnosed
with a rectal STI at the initial study visit, we did not in-
clude these infections in the analysis. Abstract Background: Exploring causal associations in HIV research requires careful consideration of numerous
epidemiologic limitations. First, a primary cause of HIV, unprotected anal intercourse (UAI), is time-varying and, if it
is also associated with an exposure of interest, may be on a confounding path. Second, HIV is a rare outcome, even
in high-risk populations. Finally, for most causal, non-preventive exposures, a randomized trial is impossible. In order
to address these limitations and provide a practical illustration of efficient statistical control via propensity-score
weighting, we examine the causal association between rectal STI and HIV acquisition in the InvolveMENt study, a
cohort of Atlanta-area men who have sex with men (MSM). We hypothesized that, after controlling for potentially
confounding behavioral and demographic factors, the significant STI-HIV association would attenuate, but yield an
estimate of the causal effect. Methods: The exposure of interest was incident rectal gonorrhea or chlamydia infection; the outcome was incident
HIV infection. To adjust for behavioral confounding, while accounting for limited HIV infections, we used an inverse
probability of treatment weighted (IPTW) Cox proportional hazards (PH) model for incident HIV. Weights were
derived from propensity score modeling of the probability of incident rectal STI as a function of potential
confounders, including UAI in the interval of rectal STI acquisition/censoring. Results: Of 556 HIV-negative MSM at baseline, 552 (99%) men were included in this analysis. 79 men were
diagnosed with an incident rectal STI and 26 with HIV. 6 HIV-infected men were previously diagnosed with a rectal
STI. In unadjusted analysis, incident rectal STI was significantly associated with subsequent incident HIV (HR (95%CI):
3.6 (1.4-9.2)). In the final weighted and adjusted model, the association was attenuated and more precise (HR (95% CI):
2.7 (1.2-6.4)). Conclusions: We found that, controlling for time-varying risk behaviors and time-invariant demographic factors,
diagnosis with HIV was significantly associated with prior diagnosis of rectal CT or GC. Our analysis lends
support to the causal effect of incident rectal STI on HIV diagnosis and provides a framework for similar
analyses of HIV incidence. Keywords: STI, HIV, Propensity scores, Survival analysis, Men who have sex with men, Marginal structural models Page 2 of 9 Vaughan et al. BMC Medical Research Methodology (2015) 15:25 Page 2 of 9 Background longitudinal measures of confounders (i.e. high-risk sex). This report serves as an applied methodological exposition
of propensity score weighting that pairs with a forthcoming
clinically-oriented report on the associations between a
broader set of STIs (including urethral bacterial STIs and
syphilis) and HIV [14]. We hypothesized that, controlling
for time-varying and time-invariant behavioral and demo-
graphic factors, the positive association between rectal STI
and subsequent HIV infection would be mitigated, yielding
an estimate of the causal effect. g
As with many preventive exposures, causal associations be-
tween preventive exposures (e.g. pharmaceuticals) and inci-
dent HIV may be optimally assessed through randomized
clinical trials (RCT). However, causal associations between
non-preventive exposures, such as high-risk sexual or sub-
stance use behaviors, and incident HIV cannot be ethically
evaluated using an RCT. Additional limitations may further
increase the analytic complexities of assessing these causal
relationships. First, high-risk sex behaviors, including un-
protected anal intercourse (UAI), receptive anal intercourse
(RAI), and partners selected from a high HIV prevalence
pool, are time-varying and, as necessary causes, must occur
in an interval prior to HIV diagnosis [1]. Given an HIV-
related exposure of interest that similarly requires high-risk
sex risk behaviors (such as anal trauma or another sexually
transmitted infection), these behaviors must be modeled as
time-varying factors that may be on a confounding path
[2]. Additionally, regression-based incidence analyses are
further challenged by limited statistical power due to rela-
tively small numbers of incident HIV, even among high-risk
populations. Recent studies have found annual HIV inci-
dence of MSM in urban areas of the United States of 1-7%,
requiring large cohorts observed for long periods of time to
accumulate sufficient events for analysis [3-7]. Common support assessment The degree to which the propensity score has been appro-
priately specified may initially be ascertained through
evaluation of common support. Common support is de-
fined by overlapping distributions of propensity scores
between exposure groups. Unlike an RCT, confounding in
an observational study will almost certainly lead to differ-
ent distributions of propensity scores between exposure
groups. However, overlap in the distributions indicates the
potential for a member of the exposed group to be in the
unexposed group and that individuals with each level of
covariates may have either exposure status (i.e. supporting
the assumptions of exchangeability and positivity) [23]. A
lack of common support, or a complete separation of pro-
pensity scores between the exposure groups indicates
severe differences between the two exposure groups and
the possibility that confounding cannot be reduced using
propensity methods [22]. Data sources and definitions For individuals
with incident STI, person-time was calculated as the dif-
ference between the date of STI diagnosis and the date
of HIV seroconversion or censoring; for individuals with-
out an STI, person-time was calculated as the difference
between the enrollment date and the date of HIV sero-
conversion or censoring due to study completion or loss An important example of these analytic limitations is the
potential causal association between HIV and sexually
transmitted infections (STI). Although recent studies have
observed associations between these two infections, evaluat-
ing a casual association requires adequate control of high-
risk sex in the time period immediately preceding both STI
and HIV diagnosis and of patterns of high-risk behaviors
[3-7,9,10]. Additionally, sexual history in the interval prior
to each diagnosis is required to best control for the poten-
tially time-varying nature of the confounding and to ac-
count for changes in behavior that may result from rectal
STI diagnosis (Figure 1) [11-13]. Therefore, to address these
analytic requirements, we detail the application of propen-
sity score weighting to examine the causal effect of rectal
bacterial STI on HIV acquisition [8]. We use a cohort of
Atlanta-area MSM with biologically measured exposure
(i.e. incident STI) and outcome (i.e. incident HIV) data and Vaughan et al. BMC Medical Research Methodology (2015) 15:25 Page 3 of 9 Figure 1 Directed acyclic graph (DAG) illustrating the hypothesized rectal STI-HIV association and time-varying behavioral confounding. c graph (DAG) illustrating the hypothesized rectal STI-HIV association and time-varying behavioral confounding. Figure 1 Directed acyclic graph (DAG) illustrating the hypothesized rectal STI-HIV association and time-varying behavioral confounding. to follow-up. The date of HIV seroconversion was esti-
mated as halfway between the dates of the final (ie: sero-
conversion) visit and penultimate visits [18]. of behavioral confounding, these potential confounders
should temporally precede both the exposure and the out-
come and be associated with both. This model then esti-
mates probabilities of exposure conditional on a set of
measured preceding covariates, or “propensity scores”. These scores may be incorporated in subsequent covariate-
adjusted, stratified, matched, or weighted analyses in order
to balance covariates across exposure groups in an obser-
vational study or to control residual confounding resulting
from a failure of randomization [8,20]. Analysis methods A crude hazard ratio (HR) for the association between
incident rectal STI and incident HIV was calculated
using an unadjusted Cox proportional hazards (PH)
model. To adjust for behavioral confounding of the rectal STI-
HIV association, while accounting for a limited number of
incident HIV infections, we used an inverse probability of
treatment weighted (IPTW) Cox proportional hazards (PH)
model for incident HIV, where the weights were derived
from propensity score modeling of STI incidence (i.e. a mar-
ginal structural model) [19]. We note that the propensity
score literature typically employs the word ‘treatment’ to dif-
ferentiate the two exposure groups. As our exposure is not a
treatment, we use the term ‘exposure groups’ rather than
‘treatment
groups’. We
first
conceptually
outline
the
approach as a four-step process and then detail the specific
application to the rectal STI-HIV association. In time-to-event analyses, applying propensity scores
using inverse probability of treatment weights (IPTW)
minimizes bias relative to the other methods of applying
propensity scores [21]. IPTW are defined as the inverse
of the propensity score. In order to reduce the influence
of outlying weights (i.e. those observations with a very
high or very low propensity score), weights may be stabi-
lized via multiplication by the mean propensity score of
the given exposure group [19,22]. Weighted survival analysis
h
l
l
d
d We then calculated an adjusted HR using IPTW-weighted
Cox proportional hazards (PH) regression, modeling inci-
dent HIV as a function of the following predictors of HIV
incidence and high-prevalence sexual networks: diagnosis
with an incident rectal STI, UAI in the interval of HIV diag-
nosis/censoring (as defined above), any reported black part-
ners in the interval of HIV diagnosis/censoring, and age at
HIV diagnosis/censoring [14,18]. The proportional hazards
assumption for STI-HIV HR was assessed using visual
examination of log-log survival curves and goodness-of-fit
testing using Schoenfeld residuals [34]. IPTW-weighted
adjusted survival curves were created [35]. Propensity score estimation When applied to observational data, properly specified
propensity scores simulate the gold-standard of epidemi-
ologic studies, the RCT [8]. In an RCT, all potential con-
founders, both measured and unmeasured, are, on
average, evenly distributed across (and thus independent
of) exposure status. However, using non-randomized observational data,
as in our analysis, exposure status may be associated
with measured and unmeasured covariates. Propensity
score estimation begins by modeling exposure status
(generally using logistic regression) as a function of poten-
tial confounders of the exposure and outcome. In the case Page 4 of 9 Vaughan et al. BMC Medical Research Methodology (2015) 15:25 Common support assessment We examined common support through visual compari-
son of the distributions of modeled probabilities strati-
fied by observed rectal STI status [31]. Balance assessment The primary confounder of the STI-HIV association,
interval-specific UAI, was defined as reporting any UAI
partners, condom failure, or inconsistent condom use in
the six months prior to rectal STI diagnosis or censoring. For the interval-specific reporting of black partners, missing
data in the interval of interest were replaced by data from
the most recent interval with data. Non-injection drug use
was defined as a positive drug-screen at baseline or self-
reported drug use at any interval. Poverty was defined as
the 2006–2010 American Community Survey estimate of
the percent living in poverty for the census-tract that in-
cluded the participant’s baseline home address. Given common support, the degree to which confounding
by the modeled factors has been controlled may be
assessed by examining balance, or distribution of potential
confounders by exposure status. Balance is assessed after
applying propensity scores to the sample in a method
analogous to that use in the final exposure effect estimate
(i.e. apply IPTW to the sample before assess balance). Bal-
ance is most often examined using standardized bias, cal-
culated as the difference in mean covariate value between
exposure groups, divided by the standard deviation of the
covariate in the entire study sample following application
of propensity scores [8]. While a standardized bias <0.25 is
considered to indicate balance of the potential confounder
between exposure groups, others have proposed a thresh-
old of <0.10 [24,25]. In the absence of balanced potential
confounders following application of propensity scores, the
final effect estimate is prone to residual confounding and,
therefore, a better propensity model should be developed. Statistical testing is inappropriate for assessing balance be-
tween treatment groups because balance is a property of
the sample and not of an underlying population [26,27]. Each participant was assigned a weight defined as the
stabilized inverse propensity score. Balance assessment We examined balance of potential confounders by calculat-
ing standardized bias. In order to examine the changes in
confounder distribution due to IPTW, we calculated stan-
dardized biases for the original sample and for the sample
following application of IPTW (i.e. the selected method of
propensity score application) [31-33]. Application: estimating the rectal STI-HIV association
Propensity score estimation Using logistic regression, we modeled the probability of
exposure (incident rectal STI) as a function of the follow-
ing potential confounders: participant’s race, participant’s
age at rectal STI diagnosis/censoring, age-race interaction,
UAI in the interval of rectal STI diagnosis/censoring, any
reported black partners in the interval of rectal STI diagno-
sis/censoring, any reported receptive anal intercourse
(RAI) for the duration of the study, census-tract-level pov-
erty, diagnosis of any non-rectal STIs for the study dur-
ation, and any non-injection drug use for the duration of
the study. These covariates, chosen a priori, may confound
the association of interest as markers of individual risk
behaviors or markers of high-prevalence sexual networks,
and have been strongly associated with STI and HIV inci-
dence [18,28-30]. All analyses were performed in SAS v9.3 (SAS Institute,
Cary, NC) [36]. Code for this analysis is available in
Additional file 1. The Institutional Review Board of Emory
University approved this study. All participants provided
written informed consent prior to enrollment. Weighted survival analysis Application of propensity scores from a model exhibiting
both common support and balance will reduce or eliminate
confounding by those measured covariates. For time-to-
event analyses, application of propensity scores using IPTW
(rather than matching, stratification, or adjustment) pro-
duces effect estimates with minimal bias [21]. When applied
to a Cox PH model, IPTW creates a pseudo-population that
permits estimation of the casual effect of the exposure on
the outcome, given that all confounders were appropriately
accounted for in the propensity model. These models may
also be further adjusted by predictors of the outcome to in-
crease precision of the final effect estimate. Results Of 803 men originally enrolled in the study, 562 (70%) had
HIV-negative screening results at baseline. Six men were
found to be acutely infected with HIV at the three-month
visit, leaving 556 men who were truly HIV-negative at base-
line and enrolled prospectively. 552 (99%) men had
complete data for all covariates and were included in this Vaughan et al. BMC Medical Research Methodology (2015) 15:25 Page 5 of 9 Figure 2 Evaluation of common support using distributions of
propensity scores for each exposure group. analysis. Of these men, over the course of the study, 79
(14%) were diagnosed with an incident rectal STI and 26
(5%) men were diagnosed with HIV (Table 1). In 6 men
(23%), the incident rectal bacterial STI preceded the HIV
infection. In unadjusted analysis, incident HIV was significantly
associated with prior incident rectal bacterial STI (HR
(95% CI): 3.6 (1.4, 9.2)). As expected based on our a priori confounding assump-
tions (Figure 1), the observational nature of the data resulted
in different, but overlapping, distributions of propensity
scores between exposure groups (Figure 2). This difference
indicates the true confounding potential due to the imbal-
ance in these covariates. However, the overlapping ranges in-
dicates that the propensity model exhibits common support. Application of IPTW resulted in balanced covariates be-
tween exposure groups, including those covariates that
were strongly unbalanced in the unweighted data (Tables 2
and 3). The standardized biases for all covariates were
below the generally accepted 0.25 threshold, and, with the
exception of the standardized bias for RAI, were below the
more conservative 0.10 threshold, suggesting minimal dif-
ferences in the weighted distributions between exposure
groups following application of IPTW. Figure 2 Evaluation of common support using distributions of
propensity scores for each exposure group. of the HR following control for confounding is evident in
the crude and adjusted survival curves (Figure 3). While not all covariates in the propensity model were
significantly associated with incident rectal STI (Additional
file 2: Table S1), all were retained due to previously ob-
served associations with incident STI and HIV and their
association with high-risk sexual networks [18,28-30]. Discussion Our analysis demonstrates the use of propensity score
methods to examine the causal effect of rectal bacter-
ial STI infection on HIV seroconversion. This method
accounts for time-varying behavioral confounding and
maximizes the information obtained from a limited
number of events, permitting observational data to
simulate an RCT. We found that, in a cohort of ini-
tially HIV-negative MSM, controlling for time-varying
risk behaviors and time-invariant demographic factors, In the final weighted and adjusted model, incident rectal
STI was significantly associated with incident HIV (HR
(95% CI): 2.7 (1.2, 6.4)). Full Cox PH model results are pro-
vided in Additional file 2: Table S2. The weighted, adjusted
association was both more precise and attenuated (25% re-
duction) when compared to the crude HR. The attenuation Table 1 Characteristics of individuals with rectal STI and HIV infections among a cohort of Atlanta-area MSM
Incident rectal STI
Incident HIV
Yes (n = 79)
No (n = 473)
Yes (n = 26)
No (n = 526)
Median (IQR)
Median (IQR)
OR1 (95% CI)
Median (IQR)
Median (IQR)
OR1 (95% CI)
Age at diagnosis
25.0 (22.1, 28.5)
28.1 (24.4, 33.9)
0.7 (0.5, 0.8)
24.6 (22.5, 28.8)
27.6 (24.3, 33.5)
0.6 (0.4, 1.0)
Poverty
18.2 (9.9, 29.7)
13.8 (8.8, 25.9)
1.1 (1.0, 1.1)
18.8 (10.5, 29.7)
13.9 (8.8, 26.3)
1.1 (0.9, 1.2)
N (%)
N (%)
OR2 (95% CI)
N (%)
N (%)
OR2 (95% CI)
Black race
49 (62)
202 (43)
2.2 (1.3, 3.6)
19 (73)
232 (44)
3.4 (1.4, 8.3)
Ever reporting RAI
67 (85)
326 (69)
2.5 (1.3, 4.8)
21 (81)
372 (71)
1.7 (0.6, 4.7)
Drug use
46 (58)
254 (54)
1.2 (0.7, 1.9)
13 (50)
287 (55)
0.8 (0.4, 1.8)
Black partners3
41 (52)
182 (38)
1.7 (1.1, 2.8)
16 (62)
202 (38)
2.6 (1.1, 5.8)
Reported UAI3
59 (75)
303 (64)
1.7 (1.0, 2.8)
21 (81)
341 (65)
2.3 (0.8, 6.1)
Non-rectal STI diagnosis4
18 (23)
34 (7)
3.8 (2.0, 7.2)
0 (0)
52 (10)
–
1 individuals with rectal STI and HIV infections among a cohort of Atlanta-area MSM eristics of individuals with rectal STI and HIV infections among a cohort of Atlanta-area MSM
I
id
t
t l STI
I
id
t HIV Table 1 Characteristics of individuals with rectal STI and HIV infections among a cohort of Atla
Incident rectal STI
Incident HIV 1Unadjusted OR for a five unit increase in the given variable. 1Unadjusted OR for a five unit increase in the given variable.
2 Discussion j
2Unadjusted OR for the given variable. 3In the interval of diagnosis/censoring. 4 Vaughan et al. BMC Medical Research Methodology (2015) 15:25 Page 6 of 9 Page 6 of 9 Table 2 Distribution of continuous potential confounders by exposure status in the unweighted and weighted study
samples
Unweighted
Weighted
Variable (Exposure
group)
Mean
(95% CI)
Median
(IQR)
Range
Standardized
bias
Mean
(95% CI)
Median (IQR)
Range
Standardized
bias
Age (No STI)
29.6 (29.0, 30.2)
28.1 (24.4, 33.9)
18.2-71.6
−0.45
29.2 (28.6, 29.8)
27.5 (24.0, 32.9)
18.2-71.6
0.04
Age (STI)
26.7 (25.3, 28.1)
25.0 (22.1, 28.5)
19.1-51.4
29.5 (27.9, 31.1)
27.6 (24.5, 33.9)
19.1-51.4
Poverty (No STI)
18.4 (17.2, 19.6)
13.8 (8.8, 25.9)
0.5-73.9
0.15
18.6 (17.4, 19.9)
13.9 (8.8, 26.3)
0.5-73.9
−0.04
Poverty (STI)
20.4 (17.3, 23.4)
18.2 (9.9, 29.7)
1.5-73.9
18.1 (15.2, 20.9)
14.5 (8.5, 26.6)
1.5-73.9 the analysis and ensuring that both HIV and STI were diag-
nosed using biological methods. diagnosis with HIV was significantly associated with prior
diagnosis of rectal STI. Among our sample, the time to
HIV infection was significantly decreased in men who had
been previously diagnosed with rectal STI, compared to
those who had not (Figure 3). Our analysis accounts for
the primary time-varying confounder of the rectal STI-
HIV association, UAI in the interval of STI diagnosis or
censoring, and other time-varying and time-invariant con-
founders. Adjusting for HIV risk factors in the Cox PH
model and weighting by the stabilized propensity score at-
tenuated the association and increased its precision as
compared to the crude HR. Therefore, our analysis lends
support to the association between incident rectal STI and
HIV diagnosis being truly causal. These potentially changing patterns of behavior and their
inclusion as necessary causes of both our exposure and
outcome required the use of more complex methods. Add-
itionally, the relatively small number of seroconversions in
our cohort limited the number of covariates that could be
included in a Cox PH model [37]. The application of pro-
pensity model derived IPTW to a Cox PH model solved
these analytic challenges by permitting our observational
data to approximate a randomized trial, balancing the two
exposure groups (in our case, those with and without rectal
STI) on measured confounders [8]. Also, propensity
models permitted adjustment for a large number of con-
founders without their direct inclusion in the model. Discussion For
time-to-event analyses with a small number of events,
IPTW maximizes data available while maintaining balance
of measured covariates between exposure groups and pro-
ducing a minimally biased effect estimate [21]. Associations observed in prior studies, which did not in-
clude data on specific high-risk behaviors preceding both
STI and HIV diagnoses, could have been confounded by
deviations from “typical” behavioral patterns [4,6,7]. Prior
studies have also relied on self-reported STI diagnoses [5]. Our analysis addressed these limitations, ensuring that tem-
poral relationships between STI infection and its preceding
UAI, between HIV infection and its preceding UAI, and be-
tween STI infection and HIV infection are accounted for in Limitations While our analysis addressed many issues with prior
analyses, limitations remain. The estimation of causal Table 3 Distribution of categorical potential confounders in the weighted and unweighted study samples
Unweighted
Weighted
Variable (Exposure group)
Proportion
Standardized bias
Proportion
Standardized bias
Black race (STI)
62
0.39
47
0.02
Black race (No STI)
43
46
Reported drug use (STI)
58
0.09
55
<0.01
Reported drug use (No STI)
54
54
Reported RAI (STI)
85
0.38
79
0.18
Reported RAI (No STI)
69
71
Reported black partners1 (STI)
52
0.27
41
0.02
Reported black partners1 (No STI)
38
40
Reported UAI1 (STI)
75
0.23
61
−0.09
Reported UAI1 (No STI)
64
66
Non-rectal STI diagnosis2 (STI)
23
0.45
10
0.02
Non-rectal STI diagnosis2 (No STI)
7
10
1In the interval of diagnosis/censoring. 2Urethral GC, urethral CT or syphilis. Distribution of categorical potential confounders in the weighted and unweighted study samples Table 3 Distribution of categorical potential confounders in the weighted and unwei cal potential confounders in the weighted and unweighted study samples Vaughan et al. BMC Medical Research Methodology (2015) 15:25 Page 7 of 9 Figure 3 Unadjusted and adjusted cumulative incidence curves for incident HIV by incident rectal bacterial STI diagnosis. Figure 3 Unadjusted and adjusted cumulative incidence curves for incident HIV by incident rectal bacterial STI diagnosis. Figure 3 Unadjusted and adjusted cumulative incidence curves for incident HIV by incident rectal bacterial STI diagnosis. Figure 3 Unadjusted and adjusted cumulative incidence curves for incident HIV by incident rectal bacterial STI diagnosis. with incident HIV in this analysis (Additional file 2: Tables
S1 and S2). Since UAI is a practically necessary cause of
both infections, the odds ratio between UAI and HIV
should be infinite, yet such estimates are hardly observed
in HIV/STI research, suggesting that UAI misclassification
is universal [5,7,38-41]. Based on our data, we believe UAI
to be underreported among those infected with STI or
HIV, but the direction of misclassification among those
who are uninfected is unclear. Additional studies under-
standing misclassification of this critical HIV risk variable
in MSM are needed. effect requires no unmeasured confounders and inclu-
sion of all confounders without misclassification [19]. UAI in the interval of rectal STI diagnosis is the pri-
mary, overriding confounder of the association in that
it is the most proximal, direct behavior by which an
individual could acquire both a rectal STI and HIV. References
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were unable to explore the role of multiple STIs in HIV
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HIV H
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit 38. Jansen IAV, Geskus RB, Davidovich U, Jurriaans S, Coutinho RA, Prins M, et al. Ongoing HIV-1 transmission among men who have sex with men in
Amsterdam: a 25-year prospective cohort study. AIDS. 2011;25:493–501. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 39. Anderson C, Gallo MF, Hylton-Kong T, Steiner MJ, Hobbs MM, Macaluso M,
et al. Randomized controlled trial on the effectiveness of counseling
messages for avoiding unprotected sexual intercourse during sexually
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with the disproportionate HIV infection burden among Black men who
have sex with men in 6 U.S. cities. PLoS One. 2014;9:e87298.
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City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics
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Abstract Background Anthropogenic activities significantly contribute to the dissemination of antibiotic resistance genes
(ARGs), posing a substantial threat to humankind. The development of methods that allow robust ARG surveillance is
a long-standing challenge. Here, we use city-scale monitoring of ARGs by using two of the most promising cutting-
edge technologies, digital PCR (dPCR) and metagenomics. Methods ARG hot-spots were sampled from the urban water and wastewater distribution systems. Metagenomics
was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From
the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and
tetracycline, respectively, were monitored by dPCR and metagenomics. Results The largest relative overall ARG abundance and richness were detected in the hospital wastewater and
the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The
abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up
to three orders of magnitude compared to contigs associated to prokaryotes. By metagenomics and dPCR, a similar
abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP
input (≈125–225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000 and 18,600 copies per ng
of sewage DNA (≈105–7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point. Conclusions dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG
detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative
abundance units was not obtained here (r2 < 0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments. Conclusions dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG
detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative
abundance units was not obtained here (r2 < 0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments. Keywords Antibiotic resistance gene, Metagenomics, Digital PCR, DPCR, ARG, Bacteria, Virus, Wastewater, sul2, tetW,
Resistome Keywords Antibiotic resistance gene, Metagenomics, Digital PCR, DPCR, ARG, Bacteria, Virus, Wastewater, sul2, tetW,
Resistome © The Author(s) 2024. Abstract Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included
in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data. City-scale monitoring of antibiotic resistance
genes by digital PCR and metagenomics Lucia Maestre-Carballa1,2, Vicente Navarro-López3 and Manuel Martinez-Garcia1,2* Lucia Maestre-Carballa1,2, Vicente Navarro-López3 and Manuel Martinez-Garcia1,2* (2024) 19:16 (2024) 19:16 Environmental Microbiome Maestre-Carballa et al. Environmental Microbiome
https://doi.org/10.1186/s40793-024-00557-6 Open Access Background detecting hallmark genes (e.g. SARS-CoV-2; ISO norm
ISO/TS 5798:2022). For instance, the European Refer
ence Laboratory for Antimicrobial Resistance published
a list of available and validated primers for monitor
ing ARGs (https://www.eurl-ar.eu/). Among the differ
ent PCR techniques, digital PCR provides absolute gene
quantification and surpasses the precision of qPCR with
much higher sensitivity and precision, without the need
for a calibration curve. Recently, dPCR technologies have
been implemented to quantify the mobility of ARGs [8]. Consequently, methods for quantitatively assessing the
potential mobility of ARGs that are easily applicable and
of low cost are urgently needed, and thus the urgent need
for a conclusive global framework addressing this issue in
environmental samples is beyond dispute [8]. In modern medicine, the advent of antibiotics is one
of the most remarkable achievements, revolutionizing
the treatment of bacterial infections and saving count
less lives. However, the emergence and dissemination of
antibiotic resistant bacteria pose a grave and escalating
threat to global public health, challenging the efficacy of
antibiotics [34]. Antibiotic resistance, characterized by
the ability of bacteria to withstand the lethal effects of
antibiotics, represents a multifaceted challenge of mon
umental proportions that will result in an annual death
toll of 10 million by 2050 [1]. The main mechanisms of
resistance are: limiting uptake of a drug, modification of
a drug target, inactivation of a drug, and active efflux of a
drug [36]. These mechanisms may be native to microor
ganisms or genetically acquired from other microorgan
isms via horizontal gene transfer. Here, we employ a city-scale distribution of waterborne
ARGs by using two of the most promising cutting-edge
technologies for ARG surveillance: metagenomics and
digital PCR data from environmental samples, focusing
on genes involved in tetracycline and sulfonamide resis
tance and balancing broad coverage and high sensitivity. By using metagenomics, we first monitored the diversity
and abundance of ARGs throughout the whole water
system in Alicante city (≈331,000 people, Spain) includ
ing five sampling locations: untreated drinking water
that feeds Alicante City, wastewater from the largest
hospital, input and output from one of the largest waste
water treatment plants (WWTP) in Alicante city, and a
seawater sample taken nearby (dozens of meters) to the
mouth of the WWTP that discharges the treated waste
water to the Mediterranean Sea. In addition to monitor
ing ARGs in the prokaryotic fraction, we also considered
the viral fraction in some of the samples. Background Then, from the
list of detected ARGs in all samples (core ARGs), we sub
sequently selected the genes sul2 and tetW to be closely
monitored by digital PCR and metagenomics. These
genes confer resistance to sulfonamide and tetracycline
antibiotics respectively, and are commonly detected in
sewage and associated with the ‘farm to fork’ [44]. In
addition, the sul2 gene is highly mobilized by plasmids
[17], and for instance, tetW gene was ranked among the
top 15 ARGs most frequently found in 79 wastewater
samples analyzed from 60 different countries and 5 con
tinents [14]. Antibiotic Resistance Genes (ARGs) can emerge vir
tually anywhere in the world and can be spread through
various means, such as water, food or air, and at differ
ent transference rates [27]. ARGs have been detected in
natural environments (e.g. aquatic, soil, and air), engi
neered and clinical habitats [45], and human microbiome
[25]. Anthropogenic activities, including the clinical use
and abuse of antibiotics and farming are widely regarded
as the main drivers of ARGs dissemination [45]. Multiple
examples of ARGs, such as the New Delhi metallo-beta-
lactamase genes, have emerged clinically and rapidly dis
seminated worldwide [21]. Unfortunately, there are many
examples like that, and recently a large metagenomic
study has demonstrated that 25% of the detected ARGs
in various habitats, pose an evident health risk [45]. Unraveling the intricate web of ARGs, elucidating their
genetic architecture, dissemination mechanisms, and set
tling methods for properly monitoring ARG dispersion
are fundamental pursuits for correct global surveillance. For instance, metagenomics, and in particular sew
age metagenomics, has been proposed as a convenient
method for ARG monitoring to determine the diversity
and abundance [14, 32]. Surveillance solutions based
on shotgun metagenomic sequencing have the advan
tage of being relatively hypothesis-agnostic albeit well-
settled international metagenomic procedure standards
(i.e. experimental and bioinformatic analysis) are clearly
lacking, but under consideration in different fields [20]. To the best of our knowledge, the only example in ARG
monitoring is the recent ISO-certified bioinformatic
workflow for the identification and surveillance of ARGs
from bacterial genomic data from isolates, which was
compared and validated with PCR and quantitative PCR
[38], methodology which allows to determine the amount
of product in real-time. Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Page 2 of 12 (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome Background Undoubtedly, PCR-based sur
veillance methods have been shown to be highly robust,
useful, consistent, and universally standardized for Thus, our study represents one of the first side-by-side
comparisons of metagenomic and dPCR data from rep
resentative urban samples in line with the One Health
strategy. Although it does not aim to settle the debate
about the best strategy to follow, which requires a large
collaborative translational effort, our study provides valu
able insights that aid in discussing the pros and cons of
each technology in the real context of a medium-sized
city. Page 3 of 12 (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome Materials and methods
Sampling spotsf tetW) by dPCR in water samples from hospital, WWTP
input, WWTP output, and seawater, and the search for
the ARG presence in RNA and DNA in viruses from
waters of the WWTP input and output. Different hot spots of the city of Alicante (331,000
citizens) were sampled (Fig. 1). The following water
samples were analyzed: (1) Untreated drinking water
from one of the main water channel of Crevillente
(38°11’23.4"N, 0°58’13.5"W; 11/20/20) that feed Alicante
city, (2) Wastewater samples from the largest hospital
in Alicante (Hospital General Universitario Dr. Balmis,
38°21’47.9"N, 0°29’08.6"W; 05/14/19), (3) the inlet of
the WWTP l’Alacantí Nord, which receives municipal
wastewater, from now on “WWTP input”, (38°25’30.8"N,
0°25’10.1"W; 03/26/19, 01/28/20 and 02/19/20), (4) the
output or treated wastewater by the aforementioned
WWTP (38°25’38.5"N, 0°25’03.5"W; 05/11/16, 01/02/20
and 02/19/20) and (5) seawater obtained in a spot nearby
the WWTP outlet placed in Campello (38°25’08.6"N,
0°23’16.7"W; 02/27/19 and 11/18/20). None of the sam
pled days recorded any rainfall. Water samples from hospital (10 mL), WWTP input
(10 mL), WWTP output (10 mL), and untreated drink
ing waters (106 mL) were filtered using a 0,2 μm filter
(Isopore Membrane Filters, Ref. GTTP02500). Those fil
ters were used to perform the DNA extraction from the
prokaryotic fraction. Prokaryotic DNA fraction from
the seawater sample used in this study was obtained as
described [29]. The acid nucleic extraction was per
formed using MasterPure Complete DNA and RNA puri
fication (Epicentre, Ref. MC85200) for all prokaryotic
samples except the untreated drinking water, which was
processed with DNAeasy PowerSoil Pro (Qiagen, Ref. 47,014) as recommended by the manufacturer. WWTP
output samples were the control samples from the Mae
stre-Carballa (2019) study that were processed as indi
cated in the paper [24]. Bioinformatic analysis y
Raw data from water samples was quality-filtered using
Trimmomatic 0.36 [3] (SLIDINGWINDOW:4:20, MIN
LEN:36). Then, the filtered and clean reads were assem
bled using SPAdes [2] (-meta), and only the obtained
contigs > 500 pb were considered for further analysis. From those contigs, ORFs were predicted using Prodi
gal program [15]. ARGs were annotated by comparing
the ARG databases ARG_ANNOT [13], RESFAMS [11],
and CARD [16] with both the assembled (ORFs) and
unassembled data (reads) using blast. Only the best-hits
with a bit-score ≥ 70, e-value < 10− 5, and identities ≥ 50%
or ≥ 90% (both thresholds were initially compared select
ing later the cut-off of ≥ 90% as likely the most reliable)
were considered as potential ARG. The housekeeping
genes present in the used ARGs databases were not con
sidered in our metagenomic analysis due to the difficulty
of determining if they were bona fide ARGs using our
thresholds [25] According to the database CARD [16] or
the nr database (NCBI), the detected ARG were grouped
by the antibiotic they confer resistance to. ARG abun
dance and normalization were estimated by dividing the
total number of ARGs per Gb of metagenome (assembled
or unassembled) and if necessary, by volume sample as
well. Estimation of shared ARGs in the analyzed samples
was carried out using a Venn diagram from the bioin
formatics UGent webpage (https://bioinformatics.psb. ugent.be/cgi-bin/liste/Venn/calculate_venn.htpl). Those
contigs that presented two or more ARGs that conferred
resistance to at least two different antibiotic classes were
classified as multi-resistant. i
Free DNA from the viral samples concentrated with
Amicon ultra-15 (100 KDa; Millipore, Ref. UFC910008)
was eliminated using 1 ul of Turbo DNase I (Invitrogen,
Lituania, Ref. AM107) and 20 µL of DNase buffer at
37 °C. After 30 min, 4.22 µL of RNase were added and
both enzymes were deactivated 30 min later at 72 °C for
10 min. To ensure the proper nucleic acids liberation
and protein digestion, the sample was treated with 1%
of proteinase K (50 µg/µl, Epicentre, Ref. MPRK092) and
20 ul of TE 10X at 65 °C for one hour while shaking. The
enzyme was inactivated at 4 °C for 5 min. The protocol
Qiamp MinElute Virus Spin Kit (QIAgen, Ref. 53,704)
was used to extract the nucleic acids. Water sample processing and sequencing Regarding the viral fraction (< 0,2 μm), samples from
the input and output of the WWTP and hospital were
filtered through a 0,2 μm filter with a syringe (PES mem
brane, Millipore). The filtered elute water (used vol
umes at Supplementary Table S1) was then concentrated
with tangential ultrafiltration using Vivaflow (100 KDa; In total, 10 water samples were analysed to study the
ARG presence in the prokaryotic and viral fractions
(Supplementary Table S1). Three different analyses were
performed: in silico characterization of the ARG using
metagenomics, the study of two selected ARGs (sul2 and Fig. 1 Sampling locations in Alicante city used in this study. Water samples were collected from various locations in the city of Alicante considered
as hot spots for ARGs dispersion. Water samples were obtained from untreated drinking water of the Crevillente’s channel (38°11’23.4"N, 0°58’13.5"W),
wastewater of the Hospital General Universitario Dr. Balmis, (38°21’47.9"N, 0°29’08.6"W) and from the wastewater treatment plant (WWTP) l’Alacantí Nord
(38°25’38.5"N, 0°25’03.5"W) and seawater (38°25’08.6"N, 0°23’16.7"W) near the outlet of the mentioned WWTP. Picture obtained from Google Maps. Scale
bar: 5 km Fig. 1 Sampling locations in Alicante city used in this study. Water samples were collected from various locations in the city of Alicante considered
as hot spots for ARGs dispersion. Water samples were obtained from untreated drinking water of the Crevillente’s channel (38°11’23.4"N, 0°58’13.5"W),
wastewater of the Hospital General Universitario Dr. Balmis, (38°21’47.9"N, 0°29’08.6"W) and from the wastewater treatment plant (WWTP) l’Alacantí Nord
(38°25’38.5"N, 0°25’03.5"W) and seawater (38°25’08.6"N, 0°23’16.7"W) near the outlet of the mentioned WWTP. Picture obtained from Google Maps. Scale
bar: 5 km Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Page 4 of 12 Maestre-Carballa et al. Environmental Microbiome sequenced in a HiSeq X sequencer (2 × 150). Sequenc
ing was performed by Macrogen company (Seul, Rep. of
Korea). Sartorius, Ref. VF20P4) until a final volume of 19 mL,
which was again filtered by 0,2 μm as above. The filtered
sample was concentrated using Amicon ultra-15 (100
KDa; Millipore, Ref. UFC910008) to a final volume of
200 µL. For the viral ARN samples, 5 L of WWTP input
and 5 L of WWTP output were sampled. Both were cen
trifuged at 10,000 g for 20 min (4 °C) and the pellet was
discarded. The supernatant free of cells was ultracon
centrated employing Vivaflow (100 KDa; Sartorius, Ref. Water sample processing and sequencing VF20P4) until a final volume of 30 mL. 3% beef extract
(Sigma-Aldrich, Ref. B4888-50G) and NaNO3 (2 M final
concentration; Scharlau, Ref. SO05010500) were added
and the pH was adjusted to 5,5. The mix was incubated
for 30 min at room temperature and the pellet was elimi
nated after 10 min of centrifugation (2500 g). The super
natant pH was then adjusted to 7.5. Polyethylene glycol
6000 (PEG; Sigma-Aldrich, Ref. 81253-250G) at 15% and
NaCl (2%; Fisher, Ref. BB358-1) were added to precipitate
the viruses and the mix was incubated at 4 °C overnight. Viruses were obtained in the pellet after centrifugation
(10,000 g, 30 min, 4 °C) and resuspended in 10 mL of PBS
(pH 7.4) (Adriaenssens et al., 2018). SYBR Gold (Ther
moFisher Scientific, Ref. S11494) was used to confirm the
lack of bacteria in viral fraction samples, and then they
were concentrated with Amicon ultra-15 (100 KDa; Mil
lipore, Ref. UFC910008) until a final volume of 200 µL. Metagenomic RNA libraries from the prokaryotic frac
tion were performed with Illumina stranded total RNA
Prep, in which a step of rRNA depletion was included
(Ribo-Zero Plus; Illumina, Ref. 20,040,525). For the RNA
viral fraction, the metagenomic library was done using
TruSeq Stranded mRNA kit (Illumina, Ref. 20,020,594)
avoiding the step where mRNA is purified, allowing us to
analyse all RNA present in the sample. All RNA samples
were sequenced in a HiSeq 2500 (2 × 125). Sequencing
was performed at the Genomics Center of CRG (Barce
lona, Spain). Bioinformatic analysis To identify viral RNA contigs, the program hmmsearch
(hmmer.org) was used to search for RNA-dependent
RNA polymerase (RdRP) profiles. The RdRP hidden Mar
kov models (HMMs) used were downloaded from Pfam
[31], from other RNA viruses’ papers [5, 42], or gener
ated by HMMER 3.2.2 (hmmer.org) using the sequences
obtained from IMG/VR [37]. Primers were checked with a PCR reaction with the
same samples that were used to design them. The reac
tion mixture included 18,15 µL mili-Q water, 1 µL of
each primer (10 µM), 0,75 µL of MgCl2 (50 Mm; Invit
rogen, Ref. Y02016), 2,5 µL of Buffer 10x (Invitrogen,
Ref. Y020228), 0,5 µL of dNTPs 10 mM (Thermo Fisher
Scientific, Ref. 10,297,018), 0,1 µL of Taq polymerase
(Thermo Fisher Scientific, Ref. 10,342,020) and 1 µL of
sample. PCR reaction conditions were: 94 °C for 5 min,
35 cycles of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for
a minute and a half. A final extension step was included
at 72 °C for two minutes and then held at 4 °C. The PCR
products were observed with an electrophoresis gel
(Agarose 2%, TBE 1X) and sequenced by Sanger (ABI
PRISM 310 Genetic Analyzer. Applied Biosystems) in the
Research Technical Services of the University of Alicante. To validate our results regarding ARG in viral contigs,
we analysed the IMG/VR v4 database, which is the most
comprehensive database to date and contains high confi
dence viral contigs from DNA and RNA [6], and also the
viral dataset of Atlantic Ocean RNA viruses [40]. Then,
we used the same pipeline described above to study the
presence of ARG. The taxonomy, host and environment
associated to each virus were extracted from the same
database [6]. We sought to compare the relative frequency of ARGs
(unassembled data; grouped by the drug class they con
fer resistance to) with the human antibiotic consump
tion in our country region using Spearman’s correlation
coefficient and its significance with a t-student test using
R program v. 4.1.2 (R Core Team, 2007). The antibiotic
consumption data for the public hospitals and the whole
community (including public hospitals and the private
sector) was obtained from the PRAN webpage (Spanish
Action Plan on Antimicrobial Resistance; https://www. resistenciaantibioticos.es/es/publicaciones/spanish-
action-plan-antimicrobial-resistance) as defined daily
doses (DDD) of common antibiotics per 1000 inhabitants
and day from the Comunidad Valenciana region (year
2019). Bioinformatic analysis For the RNA sam
ples, instead of the RNA carrier provided in the kit, 21.25
µL of glycogen was added (20 mg/mL; Thermo Scientific,
Ref. R0551) to 200 µL of the AL buffer. DNA and RNA
concentrations were measured with Qubit HS dsDNA
(Thermo Fisher Scientific, Ref. Q32854) and HS RNA
(Thermo Fisher Scientific, Ref. Q32852) respectively. In the RNA samples, the DNA was digested with Turbo
DNase I (Invitrogen, Lithuania, Ref. AM107) for 45 min
at 37 °C to increase the ratio RNA:DNA in the sequenc
ing process. i
In addition to the physical separation of prokaryotic
(> 0,2 μm) and viral fractions (< 0,2 μm), bioinformatics
was also used to specifically detect viral contigs as fol
lows. All assembled metagenomes were analysed with
VirSorter2 [12], which identified viral contigs (max. score ≥ 0.9) in the > 0,2 μm fraction and < 0,2 μm fraction,
that were grouped in the hereafter named “putative viral
fraction”. Contigs without detecting viral proteins were Metagenomic DNA library preps were carried out with
Nextera XT DNA library kit (Illumina, Ref. FC-131-1024)
according to the manufacturer´s protocol. All samples
were sequenced in a MiSeq Illumina sequencer (2 × 300),
except the untreated drinking water sample that was Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Page 5 of 12 (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 v.7.222 [19] available in Genious v. 9.1.3 program. This
program generated the consensus sequences, which were
used as target sequences to design the sul2 and tetW
primers and dPCR probes. grouped in the prokaryotic fraction, whose origin could
be DNase-resistant DNA, DNA fragments in vesicles, or
viruses that were not detected [24]. The contigs in which
at least one ARG was detected (through blastp analy
sis of Open Reading Frames against ARGs databases, as
explained above) were annotated using Kaiju [30]. The
annotation involved a comparison with the database nr_
euk (-E 0.00001, greedy mode, 12/22/23). The primer specificity was checked using primer-Blast
(NCBI) against the nr database (NCBI). The primer
sequences were tetW_F (5’->3’) TCCAGTGGCACAGA
TGTAAAG and tetW_R (5’->3’) CTTTAGCGGAGATC
ACCAAGAT. Regarding sul2, the sequences were sul2_F
(5’->3’) ATGCGCGCGTCAAAGAA and sul2_R (5’->3’)
ATCTGCCAAACTCGTCGTTATG. Probe sequences
were for sul2 5’/6-FAM/CG CAA TGT G/ZEN/A TCC
ATG ATG TCG CC/3IABkFQ/3’ and for tetW 5’/6-
FAM/AG GTG TAC C/ZEN/G CTC TTT GGC TGT
TT/3IABkFQ/3’. Bioinformatic analysis Digital PCR for sul2 and tetW were performed in a 14,5
µL mixture reaction that included 7,25 µL of MasterMix
QuantStudio 3D DIGITAL PCR V2 MMX (ThermoFisher
Scientific, Ref. APPA26316), 4,14 µL of mili-Q water, 0,63
µL of each primer (10 µM), 0,35 µL of the probe (10 µM),
2,5 µL of MgCl2 (50 Mm) and 1 µL of the water sample or
1 µL of mQ water for the negative control. The dPCR mix was loaded into a chip QuantStudio 3D
DCPR V2 20 K CHIP (12-PACK, ThermoFisher Scien
tific, Ref. A26316). The dPCR conditions were: 95 °C for
10 min, 30 cycles of 95 °C for 20 s, 55 °C for 45 s, and
60 °C for a minute. Another phase of 60 °C for 2 min and
held at 4 °C. Chips were incubated in dark conditions
and room temperature before reading them with Quant
Studio™ 3D Digital PCR Instrument (ThermoFisher Sci
entific, Ref. 4,489,084). The obtained data were analysed
with QuantStudio™ 3D AnalysisSuite™ software (Ther
moFisher Scientific). The quantification of each ARG was
calculated by dividing the number of copies of tetW or
sul2 by the ng of DNA from the sample obtained. Rep
licates and serial dilution DNA samples were included. Before the dPCR, a qPCR was conducted with the same
conditions as the dPCR, and the product was run in an Digital PCR for tetW andsul2 APPA26316, 1 µL of each primer (10 µM), 0,75 µL of
MgCl2 (50 Mm; Invitrogen, Ref. Y02016) and 1 µL of the
dPCR product). Finally, the PCR product wads analyzed
in an electrophoresis gel (TBE, 2% agarose; Fig. 2).hi To verify the proper ARG amplification during the
dPCR reaction, the obtained dPCR product was later
recovered from the amplified dPCR chip and used as a
template in a PCR reaction with the same conditions as
above for the dPCR (21,25 µL of MasterMix QuantStudio
3D DIGITAL PCR V2 MMX, ThermoFisher Scientific,
Ref. APPA26316, 1 µL of each primer (10 µM), 0,75 µL of
MgCl2 (50 Mm; Invitrogen, Ref. Y02016) and 1 µL of the
dPCR product). Finally, the PCR product wads analyzed
in an electrophoresis gel (TBE, 2% agarose; Fig. 2). The comparison of the quantification through dPCR
and metagenomics for both ARG was performed by
contrasting the number of copies of each ARG per ng of
DNA with the number of ARG hits (identity ≥ 90%, bit-
score ≥ 70 and e-value ≤ 10− 5 with the ARG databases)
for both assembled and unassembled data. Digital PCR for tetW andsul2 Absolute abundances of two abundant and ubiquitous
antibiotic resistance genes (sul2 and tetW) in the ana
lyzed water samples were studied by dPCR. For dPCR
primers and probes design, PrimerQuest Tool (https://
eu.idtdna.com/pages/tools/primerquest) was used. To
obtain the target sequences for both ARGs, first all hits
obtained from the assembled data were clustered (95%
identity) with CD-HIT program using default param
eters. TetW and sul2 clusters were selected, and con
tigs containing one of those ARG were aligned with
each other and their corresponding ARG entry in the
ARG databases (gb|AAL59753.1|ARO:3,000,412|sul2 or
AJ222769_gene_p01 for tetW) using MAFFT Alignment Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Page 6 of 12 Page 6 of 12 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 encompassing samples from untreated drinking water
supplying Alicante city, various sewage and wastewa
ter samples, and seawater collected near the discharge
point of treated wastewater from one of the largest
WWTPs (Fig. 1). The largest ARG abundance and rich
ness (i.e. thousands of ARGs per Gb of metagenome)
were detected in hospital wastewater and WWTP input
(Fig. 3; Supplementary Table S2) with a large diversity of
ARGs conferring resistance to multiple common antibi
otics (e.g. multi-drug, beta-lactamases or macrolide-lin
cosamide-streptogramin; Fig. 4 and Supplementary Figs. S1 and S2). Although metagenomics identified common
antibiotic-resistant and multiresistant bacteria, a large
fraction remained unclassified or ambiguous at the genus
level (Fig. 4C), suggesting a large diversity of uncultured
and environmental bacteria yet to be discovered host
ing ARGs. The lowest richness and ARG abundance
-one order of magnitude lower than hospital sewage
sample- was found for the untreated drinking water,
treated wastewater, and seawater (Fig. 3). Remarkably,
the analyzed viral fractions, either DNA or RNA viruses
(hospital wastewater and WWTP input and output), did
not seem to represent a major threat for ARG dispersion electrophoresis gel (TBE, 2% agarose) to check the prim
er’s performance and accuracy of the primers and probes. electrophoresis gel (TBE, 2% agarose) to check the prim
er’s performance and accuracy of the primers and probes. To verify the proper ARG amplification during the
dPCR reaction, the obtained dPCR product was later
recovered from the amplified dPCR chip and used as a
template in a PCR reaction with the same conditions as
above for the dPCR (21,25 µL of MasterMix QuantStudio
3D DIGITAL PCR V2 MMX, ThermoFisher Scientific,
Ref. City-scale resistome First, resistome analysis through metagenomics was
conducted on a city-scale water distribution system, Fig. 2 dPCR of tetW and sul2 antibiotic resistance genes. dPCR primers and probes designed for the ARGs tetW and sul2 were first tested by qPCR (same
conditions as the dPCR) in the Hospital wastewater sample, and the PCR products were observed in an electrophoresis gel. Then, a chip-based dPCR
was run for both genes and different water samples. Only dPCR results with precision ≤ 10% were considered for our results as recommended for dPCR
standards, similarly to qPCR best practices. To verify the proper amplification of the dPCR product, a second PCR (nested PCR) was run with the primers,
and the product was analyzed by gel electrophoresis (TBE, 2%) to ensure the expected size of dPCR product. Ladder used: Gene Ruler 1 kb+. This confirms
that the detection signal obtained from probes using during dPCR was fully specific as shown in the histogram and dot plot of dPCR detection of targeted
genes (blue color) Fig. 2 dPCR of tetW and sul2 antibiotic resistance genes. dPCR primers and probes designed for the ARGs tetW and sul2 were first tested by qPCR (same
conditions as the dPCR) in the Hospital wastewater sample, and the PCR products were observed in an electrophoresis gel. Then, a chip-based dPCR
was run for both genes and different water samples. Only dPCR results with precision ≤ 10% were considered for our results as recommended for dPCR
standards, similarly to qPCR best practices. To verify the proper amplification of the dPCR product, a second PCR (nested PCR) was run with the primers,
and the product was analyzed by gel electrophoresis (TBE, 2%) to ensure the expected size of dPCR product. Ladder used: Gene Ruler 1 kb+. This confirms
that the detection signal obtained from probes using during dPCR was fully specific as shown in the histogram and dot plot of dPCR detection of targeted
genes (blue color) Page 7 of 12 Page 7 of 12 (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome Fig. 3 Relative abundance of ARG obtained by metagenomics in the different water samples of Alicante city. The abundance of ARG in each water sam
ple was studied through metagenomics for both unassembled and assembled data. Hospital wastewater and WWTP input had the highest abundance
of ARGs. City-scale resistome For the assembled fraction, all contigs from the < 0,2 μm and ≥ 0,2 μm fraction in each sample that were classified as viral according to VirSorter
(see methods), were grouped in the category putative viral fraction Fig. 3 Relative abundance of ARG obtained by metagenomics in the different water samples of Alicante city. The abundance of ARG in each water sam
ple was studied through metagenomics for both unassembled and assembled data. Hospital wastewater and WWTP input had the highest abundance
of ARGs. For the assembled fraction, all contigs from the < 0,2 μm and ≥ 0,2 μm fraction in each sample that were classified as viral according to VirSorter
(see methods), were grouped in the category putative viral fraction Fig. 4 ARGs distribution, relative abundance, and antibiotic resitant bacteria in the different water samples of Alicante. Number of ARGs shared by the
different water samples: seawater (blue), Hospital (red), WWTP input (green), WWTP output (yellow), and untreated drinking water (brown) (A). For as
sembled data, different categories of ARG (conferring resistance to multidrug, MLS, tetracycline, and aminoglycoside) were found to be more frequent in
Alicante’s water samples at two different protein identity thresholds (≥ 50 and ≥ 90%) (B). For assembled contigs, identification of the most abundant bac
teria (> 1%) present in the waters of Alicante that had (at least) one ARG that belonged to the most common categories found in the waters of Alicante (C) Fig. 4 ARGs distribution, relative abundance, and antibiotic resitant bacteria in the different water samples of Alicante. Number of ARGs shared by the
different water samples: seawater (blue), Hospital (red), WWTP input (green), WWTP output (yellow), and untreated drinking water (brown) (A). For as
sembled data, different categories of ARG (conferring resistance to multidrug, MLS, tetracycline, and aminoglycoside) were found to be more frequent in
Alicante’s water samples at two different protein identity thresholds (≥ 50 and ≥ 90%) (B). For assembled contigs, identification of the most abundant bac
teria (> 1%) present in the waters of Alicante that had (at least) one ARG that belonged to the most common categories found in the waters of Alicante (C) Fig. 4 ARGs distribution, relative abundance, and antibiotic resitant bacteria in the different water samples of Alicante. Number of ARGs shared by the
different water samples: seawater (blue), Hospital (red), WWTP input (green), WWTP output (yellow), and untreated drinking water (brown) (A). City-scale resistome For as
sembled data, different categories of ARG (conferring resistance to multidrug, MLS, tetracycline, and aminoglycoside) were found to be more frequent in
Alicante’s water samples at two different protein identity thresholds (≥ 50 and ≥ 90%) (B). For assembled contigs, identification of the most abundant bac
teria (> 1%) present in the waters of Alicante that had (at least) one ARG that belonged to the most common categories found in the waters of Alicante (C) Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Page 8 of 12 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 S4). When conversing these values into absolute number
of gene copies of sul2 and tetW per mL of sample (r2 cor
relation of 0.89–0.9; Supplementary Fig. S4), overall data
ranged from hundred thousand or thousands copy genes
per mL in sewage (maximum value of 6.86 × 107 copies/
mL for tetW gene from the WWTP input sample) up
to less than 5 copies per mL in seawater for both genes
(Supplementary Fig. S5; Supplementary Table S4). The
absolute abundance after wastewater treatment was still
high for sul2 gene, being carried by different genera, such
as Bifidobacterium and Novosphingobium (Fig. 5B). How
ever, as shown in Fig. 5B, these ARG were not detected in
any marine bacteria. S4). When conversing these values into absolute number
of gene copies of sul2 and tetW per mL of sample (r2 cor
relation of 0.89–0.9; Supplementary Fig. S4), overall data
ranged from hundred thousand or thousands copy genes
per mL in sewage (maximum value of 6.86 × 107 copies/
mL for tetW gene from the WWTP input sample) up
to less than 5 copies per mL in seawater for both genes
(Supplementary Fig. S5; Supplementary Table S4). The
absolute abundance after wastewater treatment was still
high for sul2 gene, being carried by different genera, such
as Bifidobacterium and Novosphingobium (Fig. 5B). How
ever, as shown in Fig. 5B, these ARG were not detected in
any marine bacteria. (Fig. 3 and S3) since the detection of bona fide viral con
tigs hosting ARGs (identity ≥ 90%, bit-score ≥ 70 and
e-value ≤ 10− 5) was extremely infrequent with up to three
order of magnitude lower ARG abundance than the sew
age prokaryotic fraction. City-scale resistome Finally, the antibiotic consump
tion DDD of common antibiotics per 1000 inhabitants
and day did not show a correlation with the ARG relative
frequency found in the unassembled data of Alicante’s
waters (p-value > 0.5; Supplementary Table S3). (Fig. 3 and S3) since the detection of bona fide viral con
tigs hosting ARGs (identity ≥ 90%, bit-score ≥ 70 and
e-value ≤ 10− 5) was extremely infrequent with up to three
order of magnitude lower ARG abundance than the sew
age prokaryotic fraction. Finally, the antibiotic consump
tion DDD of common antibiotics per 1000 inhabitants
and day did not show a correlation with the ARG relative
frequency found in the unassembled data of Alicante’s
waters (p-value > 0.5; Supplementary Table S3). Digital PCR vs. metagenomics: surveillance and monitoring
of two global ARGs dispersed throughout water systemf Environmental Microbiome (2024) 19:16 Page 9 of 12 Page 9 of 12 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 dPCR seawater data. This only highlights that metage
nomic assembly is imperfect [23]. Some discrepancies
were observed with dPCR data, since the highest abun
dance obtained by metagenomics for sul2 and tetW was
observed for the hospital sewage (Supplementary Table
S4), while by dPCR, maximum values were obtained for
the WWTP input sample instead (Fig. 5). Although a
similar abundance trend was observed between samples
from both approaches, direct conversion and correlation
of dPCR absolute abundance units (gene copies/ng or
mL of sample) into metagenomic abundance units (no. of
ARG hits from assembled or unassembled data) showed
r2 values below 0.4, suggesting that several intrinsic fac
tors from each technology might introduce variability
that precludes a robust correlation.hi exactly the opposite. This example likely illustrates the
advantages and disadvantages of each technique, since
metagenomics provides a broad perspective regardless
of mutation rate and diversity of ARG; as long as the tar
geted ARGs meet the applied bioinformatic thresholds. In contrast, dPCR, like any other PCR based technolo
gies, is more limited by primer specificity, and likely in
our study, primers failed to capture some part of the tetW
and sul2 genes diversity present in the hospital waste
water (see for instance Supplementary Fig. S6), which
showed the highest ARG richness and diversity (Supple
mentary Fig. 4A). y
g
In the case of metagenomics, detection of ARG from
both assembled and unassembled data analyses was per
formed considering only very strict thresholds (e.g. ≥90%
nucleotide sequence identity; see methods), such as the
one recently proposed in the ISO-certified genomics
workflow, in which cut-offs of ≥90% nucleotide sequence
identity representing “exact” or “close matches” were
considered and subsequently PCR validated [38]. To date,
in general, there is a paucity of highly accurate, repro
ducible, and standardized bioinformatic tools for ARGs
detection, and this is one of the main limiting factors for
wider application of metagenomics. In our case, we have
used powerful programs well implemented in metage
nomic analysis that rely on sequence search similarity
[25, 30]. In our study, we have used multiple well-curated
and standard ARG databases for a comprehensive analy
sis widely used in ARG surveillance (see methods), such
as the one used in the recently reported ISO-certified
genomics workflow [38]. Digital PCR vs. metagenomics: surveillance and monitoring
of two global ARGs dispersed throughout water systemf We envisage that a large trans
national effort should be executed and independently
performed by several laboratories and institutions for a
major cross-comparison of metagenomics and quantita
tive and digital PCR data that will aid assist policymak
ers and private companies in making better decisions on
how to best approach ARG surveillance. In particular, the
most interesting and challenging idea in ARG monitoring
within the One-Heath perspective would be to develop
a standardized methodology of metagenomic and dPCR
(or qPCR) in which we would be able to make a direct
conversion or correlation of relative abundance units
obtained either from assembled or unassembled metage
nomic data with absolute abundance units normalized by
extracted DNA or volume sample commonly obtained
from dPCR. In our study, as shown in the result section,
the different methodological biases introduced in each
step from each one of the methodologies likely preclude
obtaining a good correlation. The most commonly resistant identified bacteria host
ing the studied tetW and sul2 genes in sewage and treated
wastewater were overall Escherichia spp., Acinetobacter
spp., Novosphingobium spp., Bifidobacterium spp., and an
uncultured Pseduoflavonifractor spp. (Fig. 5). However,
particularly for the hospital wastewater, a large fraction
of prokaryotes hosting the gene tetW remained uniden
tified. Remarkably, in the seawater sample collected near
the WWTP discharging point, we indeed detected an
uncultured cyanobacterium hosting tetW, which suggests
a possible horizontal gene transfer event. Digital PCR vs. metagenomics: surveillance and monitoring
of two global ARGs dispersed throughout water systemf of two global ARGs dispersed throughout water system
According to the global resistome analysis, 15 different
ARGs were common and present in all the analyzed sam
ples at the city-scale level (Fig. 4A). Amongst the list of
ARG city-core, genes sul2 and tetW were selected based
on their importance and worldwide distribution [14, 44]
for side-by-side comparison by digital PCR and metage
nomics (Fig. 5). Overall, the absolute abundances of
selected genes by dPCR ranged from 6,000 to 18,600 gene
copies per ng of DNA for sewage samples (e.g. hospital
wastewater and WWTP input) to only 6.8 gene copies/
ng of DNA for the seawater sample collected nearby the
WWTP discharging point (Fig. 5; Supplementary Table Overall, a similar tendency in the abundance of sul2
and tetW from dPCR was obtained by metagenom
ics (either assembled or unassembled data; see Fig. 5
and Supplementary Table S4), with higher abundances
(36–230 ARG hits per Gb of metagenome; unassembled
data) in hospital sewage and WWTP input, and signifi
cantly lower values for the seawater sample and WWTP
output (0.06-20 ARG hits per Gb of unassembled data). When looking at the metagenomic assembled data, sul2
was not detected in the assembled contigs of the seawater
sample whereas it was detected in the unassembled and Fig. 5 Citi-scale surveillance of the ARGs tetW and sul2 by metagenomics and digital PCR. The relative and absolute abundances of tetW (blue) and sul2
(red) was studied using dPCR (gene copies per ng of DNA) and metagenomics (unassembled and assembled data; No of hits per Gb) for four water samples
obtained from the hospital, seawater, and wastewater from the WWTP input and output of the city of Alicante (A). Error bars depict standard deviations. Identification of antibiotic resistant bacteria carrying tetW or sul2 in the analyzed samples (B) Fig. 5 Citi-scale surveillance of the ARGs tetW and sul2 by metagenomics and digital PCR. The relative and absolute abundances of tetW (blue) and sul2
(red) was studied using dPCR (gene copies per ng of DNA) and metagenomics (unassembled and assembled data; No of hits per Gb) for four water samples
obtained from the hospital, seawater, and wastewater from the WWTP input and output of the city of Alicante (A). Error bars depict standard deviations. Identification of antibiotic resistant bacteria carrying tetW or sul2 in the analyzed samples (B) Maestre-Carballa et al. Conclusions One of the main goals of this study is the comparison of
metagenomic and dPCR abundance data for two of the
most globally dispersed ARGs: tetW and sul2 providing
resistance to widely used antibiotics; sulfonamides and
tetracyclines. Different hot spots at a city-scale level were
considered, and in general, both techniques were able to
show similar abundance tendencies albeit some discrep
ancies were also observed. dPCR was sensitive enough to
detect a few ARG copies and provided an accurate abso
lute estimation of gene copies per ng of DNA or analyzed
volume sample both at very high or low ARG abundance
in samples, which is a strength point for further cross-
validation studies using dPCR in ARG surveillance. In
contrast, our data showed that metagenomics provided a
broad coverage of ARG detection but was less sensitive
compared to dPCR. Some discrepancies were observed
for the ARG abundance pattern in the analyzed samples
using both methodologies. In the hospital wastewater
sample (highest ARG richness) metagenomics was likely
able to capture more ARG diversity and abundance than
the one obtained with the used, specific primers for tetW
gene in dPCR (Supplementary Fig. S6), exemplifying
the pros and cons of each technology (e.g., dPCR data is
less informative for providing the taxonomic context of
microbes carrying the targeted ARGs). Unfortunately, in
our study, a direct conversion and correlation of relative
units from metagenomics to absolute abundance units
from dPCR was not achieved, which highlights that sev
eral intrinsic methodological limitations and biases from
each one of the techniques have yet to be addressed and
preclude a robust and reliable correlation. Given that the wastewaster is a hot-spot for the ARG
dispersion [22] and considering the abundance of bacte
ria that are targeted by RNA viruses such as Pseudomo
nas preyed by Cystoviridae [26] or Escherichia coli that
could be infected by the Qβ phage [4] (being the later one
of the most prevalent antibiotic resistant bacteria identi
fied in our samples (Fig. 4B)), we sought to explore the
presence of ARGs in RNA viruses in our WWTP sam
ples. No ARGs were found in RNA viral contigs in the
analyzed wastewater samples. Discussion As the study of ARG in environmental samples has
gained more attention, several culture-independent
methods have been usually applied, such as metage
nomics, a non-targeted method that provides a broad
overview of ARG in a sample [25, 39], quantitative
PCR (qPCR) that screens specific target genes, or more
recently digital PCR, which provides some advantages
over qPCR to estimate absolute abundances of copy
genes [8, 28, 43]. Here, in a step further, we have imple
mented and compared dPCR and metagenomics to assess
the resistome and abundance of two of the most global
ARGs, representing to date likely one of the first exam
ples using in parallel two culture-independent methods. In general, both approaches revealed similar trends in
ARG abundance and were capable of detecting variations
among samples. dPCR proved to be more sensitive and
accurate than metagenomics, especially for samples with
lower ARG abundance, such as seawater. Recently, simi
lar observations were obtained in a previous survey com
paring ARG abundance by qPCR and metagenomics in
multiple environmental samples including river samples,
which showed the lowest abundances [10]. In our study,
there was a discrepancy in the ARG abundance data pat
tern, since metagenomic data indicated that ARG abun
dance was highest in the hospital wastewater followed
by WWTP input, while the results from dPCR were ARGs abundance in the input WWTP was lower than
the one found at the WWTP output, due to the WWTP
treatment, as observed elsewhere [35]. This decrease
was observed for both prokaryotic and viral fractions. A substantial portion of microbes harbouring antibiotic Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 Page 10 of 12 Maestre-Carballa et al. Environmental Microbiome (2024) 19:16 resistance genes (ARGs) remained unidentified, point
ing out to a considerable diversity of antibiotic resistant
bacteria yet to be revealed. Among the ones that could
be classified, we found many bacteria related to faecal
contamination (i.e. Escherichia coli, Enterococcus fae
cium, Campylobacter coli), a factor which could largely
explain the ARG abundance in polluted sewage envi
ronments [18]. Interestedly enough, a comprehensive
analysis of ARGs present in the IMG/VR v4 database
with over five millions of viral genome and genome frag
ments (5,621,398 high-confidence viral contigs) showed
that only ~ 0.04% of the viral contigs harbour ARG, with
being the multidrug resistance the most frequent cat
egory (Supplementary Table S5). Supplementary Information Supplementary Information Among the ARGs found in our samples, we selected
sul2 and tetW due to their importance, since both are
included in a recent reported list of ARGs that pose a
worldwide health risk [45]. Unexpectedly sul2 abundance
after treatment was still high (unlike tetW), suggesting
that a large potential of sul2 ARG could have been dis
charged into natural environments, such as the Mediter
ranean Sea. However, these ARG do not seem to be later
acquired and evolutionary selected by autochthonous
marine bacteria (Fig. 5). The only case that we found that
points out in that direction is the presence of tetW in a Discussion Most of DNA viruses
carrying at least one ARG were classified as Caudovirice
tes and Tubulavirales, and the host bacteria were in fact
human associated species (Suppl. Table S6). cyanobacteria, that could represent a case of horizontal
gene transference (HGT). The presence of tetW in cyano
bacteria has also been found using dPCR in eight water
samples nearby the Taihu Lake (China) [41]. Conclusions In order to corroborate
our data, we explored the presence of ARG in other RNA
viral datasets such the ones included at IMG/VR V4 [6],
that contains, among others, RiboV1.4 with a total of
378,253 RNA viruses obtained from the study of differ
ent metatranscriptomes [33] and also RNA viral contigs
from Atlantic ocean recently published (n = 2692) [40]. We did not find any ARG in those RNA viruses with the
thresholds used. The absence of ARG in RNA viral con
tigs could be explained by the short genome size of those
viruses [7], being unlikely to carry auxiliary metabolic
genes. Overall our data suggest that although wastewa
ter viruses could act as a reservoir of ARGs, and in good
agreement with other authors [9], they do not seem to
represent a major threat for the ARG dispersion, espe
cially after the WWTP treatment which reduces the ARG
abundance. Abbreviations Abbreviations
ARGs
Antibiotic Resistance Genes
MLS
Macrolide-Lincosamide-Streptogramin
WWTP
WasteWater Treatment Plant Abbreviations
ARGs
Antibiotic Resistance Genes
MLS
Macrolide-Lincosamide-Streptogramin
WWTP
WasteWater Treatment Plant
Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s40793-024-00557-6. Supplementary Material 1: Supplementary Figures
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d 18. Karkman A, Pärnänen K, Larsson DGJ. (2019) Fecal pollution can explain
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ronments. Nat Commun 10. Sequencing data is deposited under BioProject numbers PRJNA1029559
(water samples) and PRJNA1029899 for WWTP input and output viral samples
(RNA and DNA). 19. Katoh K, Misawa K, Kuma KI, Miyata T. MAFFT: a novel method for rapid mul
tiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–66. Acknowledgements We thank Dr. Elena Gonzales Toril for providing a Coriolis instrument for this
research. We thank Preventive Medicine Unit of Hospital de Alicante and EDAR
Alacantí Nord for providing access for water samples. Page 11 of 12 Page 11 of 12 (2024) 19:16 (2024) 19:16 Maestre-Carballa et al. Environmental Microbiome Maestre-Carballa et al. Environmental Microbiome Funding 16. Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, et al. CARD
2017: expansion and model-centric curation of the comprehensive antibiotic
resistance database. Nucleic Acids Res. 2017;45:D566–73. This project was supported with UAIND18-05 grant from the Program
“Predoctoral Training in Collaboration with Companies” by the Office of the
Vice President for Research, Development, and Innovation (University of
Alicante). Funds were also provided by the Hospital Elche Crevillente Salud SL
(ref. HOSPITALECLHE1-18Y). 17. Jiang H, Cheng H, Liang Y, Yu S, Yu T, Fang J, Zhu C. Diverse Mobile genetic
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from large markets in Zhejiang, China. Front Microbiol. 2019;10:1787. Ethics approval and consent to participate
Not applicable. 21. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and
the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 2010;10:597–602. Author contributions 14. Hendriksen RS, Munk P, Njage P, van Bunnik B, McNally L, Lukjancenko O, et
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prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11:119. Competing interests The authors declare no competing interests. 22. Kunhikannan S, Thomas CJ, Franks AE, Mahadevaiah S, Kumar S, Petrovski S. (2021) Environmental hotspots for antibiotic resistance genes. Microbiolo
gyopen 10. Received: 17 January 2024 / Accepted: 22 February 2024 Received: 17 January 2024 / Accepted: 22 February 2024 (2024) 19:16 (2024) 19:16 (2024) 19:16 44. Yang D, Heederik DJJ, Scherpenisse P, Van Gompel L, Luiken REC, Wadepohl K
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MB et al. (2023) An ISO-certified genomics workflow for identification and
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Zhang Z, Zhang Q, Wang T, Xu N, Lu T, Hong W et al. (2022) Assessment of
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and total virioplankton mass contribution from surface to the deep ocean. Environ Microbiol. 2023;25:3151–60. 42
Wolf YI, Silas S, Wang Y, Wu S, Bocek M, Kazlauskas D et al. (2020) Doubling of
the known set of RNA viruses by metagenomic analysis of an aquatic virome.
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Wolf YI, Silas S, Wang Y, Wu S, Bocek M, Kazlauskas D et al. (2020) Doubling of
the known set of RNA viruses by metagenomic analysis of an aquatic virome. Nat. Microbiol. 2020 510 5: 1262–1270. 43. Yang R, Paparini A, Monis P, Ryan U. Comparison of next-generation droplet
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Time series analysis of total and direct associations between high temperatures and preterm births in Detroit, Michigan
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Open access
Original research
Carina J Gronlund ,1 Alyssa J Yang,2 Kathryn C Conlon,3
Rachel S Bergmans ,4 Hien Q Le,5 Stuart A Batterman,6 Robert L Wahl,7
Lorraine Cameron,8 Marie S O'Neill9
To cite: Gronlund CJ, Yang AJ,
Conlon KC, et al. Time series
analysis of total and direct
associations between high
temperatures and preterm births
in Detroit, Michigan. BMJ Open
2020;10:e032476. doi:10.1136/
bmjopen-2019-032476
►► Prepublication history and
additional material for this
paper are available online. To
view these files, please visit
the journal online (http://dx.doi.
org/10.1136/bmjopen-2019-
032476).
Received 20 June 2019
Revised 17 December 2019
Accepted 09 January 2020
© Author(s) (or their
employer(s)) 2020. Re-use
permitted under CC BY.
Published by BMJ.
For numbered affiliations see
end of article.
Correspondence to
Dr Carina J Gronlund;
gronlund@umich.e du
Abstract
Objectives Preterm births (PTBs) represent significant
health risks, and several studies have found associations
between high outdoor temperatures and PTB. We
estimated both the total and natural direct effects
(independent of particulate matter, ozone and nitrogen
dioxide air pollutants) of the prior 2-day mean apparent
temperature (AT) on PTB. We evaluated effect modification
by maternal age, race, education, smoking status and
prenatal care.
Design and setting We obtained birth records and
meteorological data for the Detroit, Michigan, USA area,
for the warm months (May to September), 1991 to 2001.
We used a time series Poisson regression with splines
of AT, wind speed, solar radiation and citywide average
precipitation to estimate total effects. To accommodate
multiple mediators and exposure-mediator interactions, AT
inverse odds weights, predicted by meteorological and air
pollutant covariates, were added in a subsequent model to
estimate direct effects.
Results At 24.9°C relative to 18.6°C, 10.6% (95% CI:
3.8% to 17.2%) of PTBs were attributable to the total
effects of AT, and 10.4% (95% CI: 2.2% to 17.5%) to direct
effects. Relative excess risks of interaction indicated that
the risk of PTB with increasing temperature above 18.6°C
was significantly lower among black mothers and higher
among mothers less than 19, older than 30, with late or no
prenatal care and who smoked.
Conclusion This additional evidence of a direct
association between high temperature and PTB may
motivate public health interventions to reduce extreme
heat exposures among pregnant women, particularly
among those who may have enhanced vulnerability.
Introduction
Preterm births (PTBs) are defined as births
that occur before 37 gestational weeks.
Babies born prematurely are at greater risk
of infant death and impaired health, such
as cerebral palsy and impairments of cognitive development, hearing, vision, respiration and digestion. In 2016, 10% of babies
were born prematurely, with statewide rates
ranging 7.8% to 13.7% in the USA.1 Within-
state disparities in preterm birth rates can be
Strengths and limitations of this study
►► We expand on a growing literature demonstrating
total effects of temperature on preterm birth (PTB)
by examining mediation of the total temperature effect by air pollutants.
►► We did not have geographic identifiers for the study
participants at a level as fine as residential address
or indoor exposure information, and we therefore
assigned time-varying environmental exposures at
an outdoor, citywide level.
►► A low prevalence of residential air conditioning
(32%) in the study time period suggests that our
exposure assignment using outdoor exposures
alone may have more accurately reflected total daily exposures than in more recent studies in other
geographies.
►► We account for multiple mediators and potential exposure-
mediator interactions by applying
a straightforward technique — the use of inverse
odds weights — to separate the total effects of temperature on PTB from the direct effects.
►► We used methods examining potential synergistic
effects, as opposed to just examining statistical interactions, by other characteristics that confer vulnerability to PTB.
greater: from 1990 to 2010, 18% of births in
Detroit, Michigan, were PTBs.2
Certain risk factors are known to be associated with PTB, such as cigarette smoking,
alcohol use, hypertension and diabetes.1 Air
pollution is associated with PTB,3 4 and higher
ambient temperature has been proposed as a
risk factor for PTB.5 6
High ambient temperature could pose a
risk for PTB through several biological pathways, including stress and dehydration pathways.7 Several studies have evaluated possible
associations between high ambient temperature and PTB, with mixed results.5 6 Studies in
cities in Australia, Quebec, China, Belgium,
Italy, Spain and across the USA have all found
significant positive associations between
Gronlund CJ, et al. BMJ Open 2020;10:e032476. doi:10.1136/bmjopen-2019-032476
1
BMJ Open: first published as 10.1136/bmjopen-2019-032476 on 5 February 2020. Downloaded from http://bmjopen.bmj.com/ on May 20, 2024 by guest. Protected by copyright.
Time series analysis of total and direct
associations between high temperatures
and preterm births in Detroit, Michigan
Open access
Methods
Outcome variable
Birth outcome data were derived from an electronic
database of birth certificate records requested from the
Michigan Department of Health and Human Services
Division for Vital Records and Health Statistics and subsequently limited to live, singleton births that occurred
from 1 January 1991 to 31 December 2001 in ZIP codes
within 4 km of one of three air quality monitors (Allen
Park, Linwood and East 7 Mile) in and bordering Detroit,
Michigan.4 In the final data set, births were categorised as
term versus PTB, defined as births that were less than 37
gestational weeks. We limited the data set to births that
occurred May to September in order to focus on heat
exposures, for a total of 9053 births.
Identification of confounders and mediators
A directed acyclic graph (DAG) was constructed to define
the causal framework and elucidate potential mediating effects of air pollutants in the association between
2
Figure 1 Directed acyclic graph of the causal framework for
mediation of the association between apparent temperature
and preterm birth by air pollutants. U = unmeasured,
C=confounder, M=mediator, E=exposure of interest,
D=outcome. Thick solid lines known associations. Thin solid
lines indicate suspected associations. The thick dashed line
indicates the direct effect pathway being tested. The thin
dashed lines indicate the indirect effect pathways being
tested. Other primary and secondary pollutants include
particulate matter less than 10 microns in aerodynamic
diameter and nitrogen dioxide.
AT and PTB (figure 1). In this DAG, AT is the exposure of interest and PTB the outcome of interest. Given
suspected associations between PTB and precipitation23
and between PTB and vitamin D, for which one source is
solar radiation,24 as well as causal meteorological relationships between AT, solar radiation and precipitation via a
parent weather system, precipitation and solar radiation
are potential confounders of the AT-PTB association. In
this DAG, primary and secondary air pollutants are the
hypothesised mediators of the temperature-PTB association given that: (1) they have been associated with the
health outcome in other locations3 and in this data set in
the third trimester,4 (2) high temperature enhances the
rate of ground-level ozone formation and (3) power plant
emissions increase when temperatures rise to accommodate additional demand for air conditioning.25 Furthermore, the weather system is a causal parent to both AT
and the pollutants and therefore a potential confounder
given that meteorological conditions such as wind speed,
precipitation and cloud cover can affect pollutant formation and exposure as well as sunlight, which increases AT
and promotes ozone formation.26 Therefore, a model
characterising the total effect of AT would need to include
the confounders of solar radiation, wind speed and
precipitation but not include pollutants. A model characterising the direct effect of AT, independent of air pollution
mediation, would need to include these mediators. The
modelling used to characterise direct and indirect effects
is further described in the Statistical Analysis section
below.
Gronlund CJ, et al. BMJ Open 2020;10:e032476. doi:10.1136/bmjopen-2019-032476
BMJ Open: first published as 10.1136/bmjopen-2019-032476 on 5 February 2020. Downloaded from http://bmjopen.bmj.com/ on May 20, 2024 by guest. Protected by copyright.
hot temperatures within the week preceding delivery
and PTB. 5 6 8–16 High ambient temperatures in the
month of conception and the third trimester were also
positively associated with PTB in Changsha, China.17 18
However, earlier, rigorously conducted time series analyses of temperature and PTB in Germany and in London,
England, did not find associations.19 20 Likewise, in
Chicago, Illinois, Porter et al found no effect of the July
1995 heat wave on gestational length.21 Sources of heterogeneity among these effect estimates may include differences in study design, prevailing climate and regional
adaptation, population structure, exposure assessment,
critical windows of exposure considered and methods of
gestational age assessment.12
Studies often control for air pollution exposure,
although the argument has been made that treating air
pollutants as confounders of temperature-health associations is inappropriate given that high temperatures can
contribute to increased concentrations of some air pollutants, and both temperature and air pollution are affected
by sunlight.22 Differences in the manner in which air
pollutants are accounted for in the temperature-
PTB
modelling may also account for some of the heterogeneity observed in this literature.
We performed a time series analysis to investigate the
association between high apparent temperature (AT) and
PTB in the Detroit, Michigan area, estimating both the
total effects of temperature as well as the natural direct
effects. To estimate natural direct effects, we excluded
potential mediation effects by the air pollutants ozone,
particulate matter with an aerodynamic diameter of less
than 10 micrometres (PM10), and nitrogen dioxide (NO2)
using an inverse odds weighting technique. We further
examined whether the maternal risk factors of age, race,
education, smoking and level of prenatal care modified
the association between high AT and PTB.
Open access
Patient and public involvement
Patients and the public were not involved in the design or
planning of the study.
Statistical analysis
Case-crossover analysis is commonly used in studies of
PTB and temperature.12 13 34 Because PTB is not a rare
event in this particular population, the case-crossover ORs
would not approximate risk ratios. Therefore, we used a
Gronlund CJ, et al. BMJ Open 2020;10:e032476. doi:10.1136/bmjopen-2019-032476
time series design with Poisson regression, controlling for
seasonal and long-term variations in PTB counts with a
cubic b-spline for day-of-year, with 5, two or eight knots,
and a cubic b-spline for year with two knots. AT exposure was characterised as 2-day mean AT, or the mean
of the AT values on the day of and the day before birth.
For AT and the covariates, non-linearity was considered
by initially modelling each as a b-spline with three knots
and selecting a single knot for subsequent modelling of
the covariate as a piecewise linear spline when substantial non-linearity was visually evident. The 95% CIs were
constructed from the 2.5th and 97.5th percentiles of 500
bootstrapped samples. In sensitivity analyses, given its
wide usage for this research question, a time-stratified
case-crossover design was used with time strata defined as
2- or 3-week periods.
To account for missing air pollutant values, we
conducted multiple imputations using chained equations. We used a more general model including lag days
0 to 2 of the above meteorological and pollutant values.
The air pollutant values were well-predicted by lag days
0 to 2 meteorology and air pollution values, and from
examinations of trace plots, or scatter plots of successive
parameter estimates, we determined that a total of three
imputations following two burn-
ins was sufficient. All
subsequent analyses were conducted on each of the three
resulting data sets.
To estimate the total effects of AT, we included terms
for solar radiation, wind speed and precipitation in the
Poisson model, which blocked all of the paths in the DAG
from AT to PTB that did not pass through the mediators
(figure 1). However, to estimate natural direct effects,
we used a more generalisable technique — inverse odds
weighting — given that we had multiple mediators and as
well as potential exposure-mediator interactions.35 In this
technique, we first fit a standard linear regression of 2 day
mean AT (meanAT01) on the air pollutant mediators and
covariates (solar radiation, wind speed, precipitation).
We then estimated:
inverse odds = 1/exp(predicted meanAT01/σ 2 ) Equation
1
where σ2 was the model mean squared-
error. The
inverse odds were then used as weights in the subsequent
analysis of PTB and AT with the other covariates but not
the air pollutants. The weights render AT independent of
the mediators, thereby allowing the estimation of AT separately from the effects of the air pollutant mediators on
PTB. Total and direct effects were calculated from each
of the three imputed data sets and then each averaged.
To assess the public health impact of AT on PTB, we
estimated the attributable fraction (AF), or the percent
of PTB attributable to high AT among women exposed to
the high AT. The AF was estimated as: 1/(1 – RR) * 100%
where the RR (relative risk) was for the 95th percentile
(24.9°C) relative to the 50th percentile (18.6°C) of May
to September 2-day mean AT. Indirect effects were estimated as the difference between the total effects and the
3
BMJ Open: first published as 10.1136/bmjopen-2019-032476 on 5 February 2020. Downloaded from http://bmjopen.bmj.com/ on May 20, 2024 by guest. Protected by copyright.
Exposure variables
Daily mean temperature, dew point temperature and
wind speed were obtained from the National Center for
Environmental Information Integrated Surface Daily
Lite database of daily weather parameters from weather
stations worldwide.27 Data from the Detroit City Airport
was used due to its proximity to the mothers’ ZIP codes
of residence. To better represent thermal discomfort, we
used AT rather than air temperature. AT is similar to heat
index, and increases with both air temperature and relative
humidity. AT was calculated using the following formula:
(−2.653 + 0.994×(temperature in °C)+0.0153×(dew point
temperature in °C)2).2829 From Detroit City Airport, 11%
and 15% of temperatures and dew points were missing,
respectively, so data from Detroit Metropolitan Airport
were used to replace these missing data. Among non-
missing values, daily AT from Detroit City Airport was
highly correlated with that from Detroit Metropolitan
Airport (Pearson’s correlation coefficient=0.98).
The total amount of direct and diffuse solar radiation
received on a horizontal surface during each 60 min
period at the Detroit City Airport was retrieved from the
National Solar Radiation Database.30 These data were
modelled from meteorological data including cloud
cover, aerosol and ozone data from sources such as sun
photometers, satellites and albedo data.31 We further estimated daily means from the hourly solar radiation values.
Daily precipitation totals were obtained from Oregon
State University’s PRISM Climate Group.32 These are
modelled at a 4 km resolution based on observations and
a climatologically-
aided interpolation process. Rasters
were cropped to the City of Detroit and daily citywide
averages were calculated.
Daily 8 hour maximum ozone and daily mean NO2 and
PM10 concentrations were obtained from the Environmental Protection Agency for all Wayne County, Michigan monitors and averaged by day and pollutant.33 Only
daily monitor values for which at least a single daily or
18 of the 24-hourly values were available were retained,
resulting in substantial missingness, which was addressed
in the statistical analysis below.
Birth certificate data also included date of birth and
maternal age group (16 to 19, 20 to 29 and over 30), race
(black or white), smoking status (smoker vs non-smoker),
education level (less than high school vs high school or
higher) and level of prenatal care (prenatal care vs late
or no prenatal care), which were used in analyses of effect
modification.
Open access
where EM was the effect modifier of interest and the
denominator of each RR was the risk at the 50th percentile
of AT (18.6°C) and the absence of the modifier (EM=0).
RERI CIs were bootstrapped from 1000 samples. Analyses
were conducted in SAS 9.4 (Copyright 2016, SAS Institute
Inc, Cary, North Carolina, USA) using PROC GLIMMIX,
which allows for multiple splines in a Poisson regression.
Figure 2The exposure-response graph was constructed
in R (R Foundation for Statistical Computing, Vienna,
Austria) using the dlnm package37 following modelling
using the glm.nb function in the MASS package.38
Results
There were 9053 singleton PTBs in this Detroit-
area
sample, May to September, 1991 to 2001 (table 1). There
were fewer PTBs in September compared with the other
months, and consistent with Detroit’s population decline,
Figure 2 Association between mean apparent temperature
(AT) over lag days 0 to 1 and preterm birth, modelling AT as a
b-spline with 3 df.
4
Table 1 Demographics of preterm births in Detroit,
Michigan study area, May to September, 1991 to 2001
N
%
Total
Month
9053
100
May
1918
21.2
June
1944
21.5
July
1903
21.0
August
1754
19.4
September
1534
16.9
1991–1995
4708
56.2
1996–2000
3670
43.8
White
2443
27.0
Black
6433
71.1
Other
177
1.9
Non-smoker
6621
73.1
Smoker
2345
25.9
Missing
87
1.0
Prenatal care
5037
55.6
Late or no prenatal care
2960
32.7
Missing
1056
11.7
16–19 years
1782
19.7
20–29 years
4821
53.2
≥30 years
2450
27.1
Less than high school
3485
38.5
High school or higher
Missing
5451
117
60.2
1.3
Year (two equal time periods)
Maternal race
Maternal smoking status
Level of prenatal care
Maternal age
Maternal education
the number of PTBs declined with time. Considering
individual characteristics, 27.0% of the PTBs were among
white mothers and 71.1% were among black mothers. A
majority of the mothers were non-smokers (73.1%), had
prenatal care (55.6%), were 20 to 29 years of age (53.2%)
and had at least a high school education (60.2%) (table 1).
On average, AT exposure was 18.0°C, and among the
days when AT was greater than the time-period-specific
daily median of 18.6°C (ie, for the upper end of the
temperature spline), AT averaged 1.5°C higher. A total
of 53% of the cases occurred on days with no rain, so the
geometric mean precipitation was 0.4 mm. The mean
ozone concentration was 44.8 ppb and the maximum
was 102 ppb (table 2). In examining the daily time series
of ozone over the study period, the 3-year running averages of the fourth highest daily 8 hour maximum value
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direct effects. The 95% CIs were constructed from the
2.5th and 97.5th percentiles of 500 bootstrapped samples.
To understand how the effects varied among maternal
subgroups, or effect modification of the PTB-AT association, we simultaneously included interaction terms
between the AT spline and the indicator variables for black
race, age 16 to 19, age 30 years or older, low prenatal care
(late or no prenatal care), current smoker and no high
school education. We expanded the data set such that we
had rows for each unique combination of date and daily
exposures, race, age group, education, prenatal care and
smoking status. Given the large number of zero counts,
we specified a negative binomial distribution rather than
a Poisson distribution. For public health significance, we
were interested in the absolute rather than the relative
increase in PTB risk due to synergistic effects between
temperature and each modifier of interest. Therefore
we focused on additive, rather than multiplicative, interactions. Relative excess risk due to interaction (RERI)36 was
calculated for each potential modifier as:
RRAT=95th,EM=1 − RRAT=95th,EM=0 − RRAT=50th,EM=1 + 1 Equa
tion 2
Open access
N
Median
Mean
Min
Max
Two-day mean apparent temperature, °C
Two-day mean apparent temperature 18.6°C and above
9053
9053
18.6
0.1
18.0
1.5
3.1
0.0
28.0
9.4
Mean solar radiation (W/m2)
9053
226.4
217.7
38.1
352.8
Total precipitation (mm)*
9053
0.0
0.4
0.0
56.7
Mean wind speed (m/s)
9053
3.6
3.6
0.1
8.0
Maximum 8 hour average ozone (ppb)
9039
43.0
44.8
3.2
102.0
Mean particulate matter, 10 µm or less (μg/m3)*
Mean nitrogen dioxide (ppb)*
8331
8956
39.0
18.4
38.5
19.7
8.0
0.0
158.0
72.8
*Geometric means are provisded. Values were natural-log-transformed in the regression analyses.
PTB, preterm birth.
of ozone ranged from 77 to 92 ppb, suggesting that the
(current) National Ambient Air Quality Standards (70
ppb) had been exceeded in each year of the study.
In a crude model of the association between PTB and
AT where AT was modelled flexibly as a b-spline with 3
df, we found a non-linear association, with approximately
null effects below 18°C and an increasingly stronger positive association at higher temperatures (figure 2). To
address potential sensitivity of the results to control for
season, we varied the df in the day-of-year term, using 2,
5 and 8 df. The results were not highly sensitive to this
choice, with the point estimates of the percentages of PTB
attributable to AT ranging only from 8.1% (8 df) to 9.9%
(2 df) among pregnant women exposed to days when AT
was 24.9°C versus exposure on 18.6 °C days (table 3). In
case-crossover analyses, the resulting ORs were also statistically significantly greater than 1.0, regardless of whether
the time strata were 2-week, 3-week or 1-month periods
(online supplementary table 1). However, the ORs were
greater in magnitude than the risk ratios estimated in the
time series design, which is expected, given that ORs overestimate risk ratios when events are not rare.
The total effects were estimated as 10.6% (95% CI:
3.8% to 17.2%) of PTBs attributable to AT among pregnant women exposed to a day when AT was 24.9°C versus
exposure on an 18.6 °C day. Accounting for mediation
of this effect by PM10, ozone and NO2 by using inverse
odds weights, the direct effect of AT on preterm birth was
decreased to 10.4% (95% CI: 2.2% to 17.5%), although
the difference between the two values was not statistically
significant (indirect effect=0.3%, 95% CI: −1.7% to 2.6%).
In examining the RERI from interactions between
AT and maternal characteristics, black race was found
to be protective, with an RERI significantly less than 0
(table 4). The magnitudes of RERIs are not meaningful,
but this result indicates that the association between high
temperature and PTB is weaker among women of black
race than non-black race. In contrast, RERIs for age 16
to 19 years, age >30 years, low prenatal care and tobacco
smoking were all significantly greater than 0, indicating
that the association between PTB and high temperature is
stronger among women with these characteristics.
Discussion
term temperature
The strong association between short-
exposure and PTB in Detroit, Michigan, USA was consistent
with several other studies’ observed associations between
PTB or diminished gestational age and temperature exposures within the week prior to delivery at regionally high
Table 3 Relative risk of preterm birth and per cent of preterm births attributable to 2-day mean apparent temperature (AT) on
a 24.9°C day versus an 18.6°C day, Detroit, Michigan area, May to September, 1991 to 2001
Model
Knots in day-of-
year spline
Covariates
Per cent
attributable (95%
Relative risk (95% CI) CI)
1
2
2
5
None
None
1.11 (1.02 to 1.09)
1.11 (1.03 to 1.20)
9.9 (2.4 to 16.4)
9.8 (2.6 to 16.5)
3
8
None
1.09 (1.01 to 1.18)
8.1 (1.0 to 17.6)
4
5
5
5
Solar radiation, wind speed, precipitation
Solar radiation, wind speed, precipitation,
inverse-odds weights*
1.12 (1.04 to 1.21)
1.12 (1.02 to 1.21)
10.6 (3.8 to 17.2)
10.4 (2.2 to 17.5)
*Inverse-odds weights calculated from the predicted odds of AT given lag day 0 and 1 of ozone, PM10 and NO2.
NO2, nitrogen dioxide; PM10, 10 micrometres.
Gronlund CJ, et al. BMJ Open 2020;10:e032476. doi:10.1136/bmjopen-2019-032476
5
BMJ Open: first published as 10.1136/bmjopen-2019-032476 on 5 February 2020. Downloaded from http://bmjopen.bmj.com/ on May 20, 2024 by guest. Protected by copyright.
Table 2 Daily exposures among the PTB cases, Detroit, Michigan area, May to September, 1991 to 2001
Open access
Characteristic
RERI
95% CI
Black race
Age 16–19 years
−1.5
0.50
-1.9 to 1.0
0.22 to 0.76
Age >30 years
0.34
0.08 to 0.65
Low prenatal care
0.44
0.19 to 0.65
No high school
Smoker
0.19
0.52
−0.06 to 0.43
0.29 to 0.76
With five knots in the day-of-year spline.
temperatures. Regions in which these associations have
been found include Central Australia (8.3% at 40°C daily
maximum temperature),11 Northern California, USA
(11.6% increase for a 5.6°C increase in weekly average AT
in the warm season),34 Alabama, USA (32.4% increase with
two consecutive days of daily mean temperatures above
the 98th percentile),39 Barcelona, Spain (5-day reduction
in average gestational age with heat index above the 99th
percentile),8 Rome, Italy (1.9% increase per 1°C increase in
maximum AT in the prior 2 days and 19% increase during
heat waves),40 Brisbane, Australia (13% to 100% increase
during heat waves), in an aggregated sample of 12 US cities
(12% to 16% increase with 2.8°C increase in prior week),12
in Southern China (7% increase with previous-
week
temperatures above 95th percentile)10 and in a multi-city
USA sample (2% increased PTB risk with extreme heat in
the prior 4 days).41
The precise biological mechanisms by which high
ambient temperature might increase risk for PTB
are unclear, although psychosocial stress7 and dehydration pathways are plausible.7 Stress increases the
levels of cortisol and epinephrine, potentially leading
to the secretion of placental corticotropin-
releasing
hormones (CRH). Placental CRH can then activate
the fetal hypothalamic-
pituitary-
adrenal axis, which
could prompt the fetal expression of cortisol and
dehydroepiandrosterone-sulfate and placental release of
estriol and prostaglandins, potentially triggering PTB.42
Dehydration due to high temperatures and sweating
could also reduce blood flow to the uterus and induce
a greater release of antidiuretic hormone and oxytocin,
which could trigger labour onset.43
One strength of our study may be a tighter correspondence between the true ambient temperatures experienced by our sample and the measured outdoor ambient
AT given the low air conditioning prevalence in the Detroit
area during the time period. Specifically, in 1993, the last
year for which county-specific American Housing Survey
data were available in the Detroit area, only 32% of Wayne
County households had central air conditioning.44 Furthermore, central air conditioning prevalence was low in this
6
region despite a ‘hot-summer humid continental climate’
Köppen climate classification,45 allowing a fairly wide range
of warm season AT exposure over which AT-PTB associations could be examined. In contrast, regions where associations between temperature and PTB were null in rigorously
conducted daily time series studies included Brandenburg
and Saxony, Germany,20 and London, UK,19 which are
in ‘temperate oceanic climates,’ where all months have
average temperatures below 22°C.45 Additionally, Guo et al10
found an association between previous-week temperature
and PTB only in the ‘hot’ region of China, defined as having
annual average temperatures 19°C or higher. Furthermore,
in a survival analysis of previous-week temperature and PTB
in 18 European cities, the pooled effect estimates were
null, and no individual city results were presented.46 Again,
with the exception of four cities, these European cities
were all in cooler climates where all months have average
temperatures below 22 °C.45 This suggests that in regions
where the threat of extremely high temperatures is rare,
PTB is not triggered at warm temperatures, regardless of
relative temperature thresholds, even at the same absolute
temperatures at which heat-associated PTB is experienced
in warmer climates. This could be due to differences in
emotional stress47 or physiological stress responses to warm
temperatures between climates. Alternatively, the heterogeneity in effect estimates could be due to regional differences
in the misclassification of the true individually-experienced
temperatures by outdoor temperatures. Finally, in the 18-city
European study, the PTB rate was only 5%, suggesting that
PTB aetiologies may differ between Europe and the USA,
in which case the pregnancies in the European cohorts
could have been less susceptible physiologically to high
temperature.46 Other strengths of our study include our
consideration of near-surface solar radiation and weather
conditions as confounders of the total effects of AT, air
pollutants as mediators rather than confounders and estimation of the direct effects of AT as distinct from those
mediated by air pollutants. By using an analysis technique
that was ‘agnostic’ to exposure-mediator interactions and
could accommodate multiple mediators,35 we found that
most if not all of the effect of AT on PTB is direct, and not
mediated by daily changes in air pollution concentrations.
However, this mediation analysis technique tends to overestimate the CIs around the indirect effects,35 which may
account for our finding a null indirect effect of air pollutants on PTB when previous research has in fact identified
significant associations between short-term increases in air
pollution and PTB.48 We may have also underestimated
the air pollutant effects because we were only examining
the indirect effects of AT through an air pollutant pathway
rather than the total effects of air pollution and because we
only considered PM10 rather than PM2.5.
Another strength of our study is that we reported RERIs,
rather than merely reporting statistical interactions in the
models. In doing so, we provided evidence for synergistic
effects of high temperature with the independent risk
factors for PTB of prenatal care, smoking status and age.
Interestingly, in this majority-black population, the risk of
Gronlund CJ, et al. BMJ Open 2020;10:e032476. doi:10.1136/bmjopen-2019-032476
BMJ Open: first published as 10.1136/bmjopen-2019-032476 on 5 February 2020. Downloaded from http://bmjopen.bmj.com/ on May 20, 2024 by guest. Protected by copyright.
Table 4 Relative excess risk due to interaction (RERI)
for interactions of 2-day mean apparent temperature
on a 24.9°C day versus an 18.6°C day with maternal
characteristics, Detroit, Michigan area, May to September,
1991 to 2001
Open access
Author affiliations
1
Survey Research Center, University of Michigan Institute for Social Research, Ann
Arbor, Michigan, USA
2
Urban Indian Health Institute, Seattle, Washington, USA
3
Public Health Sciences, University of California Davis, Davis, California, USA
4
Psychiatry, University of Michigan Medical School, Ann Arbor, Michigan, USA
5
Toxicology and Risk Assessment, Chemours Co, Wilmington, Delaware, USA
6
Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan, USA
7
Surveillance and Program Evaluation Section, Michigan Department of Health and
Human Services, Lansing, MI, USA
8
Michigan Climate and Health Adaptation Program, Michigan Department of Health
and Human Services, Lansing, Michigan, USA
9
Environmental Health Sciences and Epidemiology, University of Michigan, Ann
Arbor, Michigan, USA
Gronlund CJ, et al. BMJ Open 2020;10:e032476. doi:10.1136/bmjopen-2019-032476
Contributors MSO, SAB, HQL, RLW and LC obtained the data or resources,
critiqued the analysis and reviewed the manuscript. RSB and KCC assisted with
preliminary analyses, critiqued the analysis and reviewed the manuscript. AJY
performed and drafted preliminary analyses and reviewed the manuscript. CJG
directed preliminary analyses, revised the analysis and revised the manuscript. We
thank Leah Comment for early stage data management and analysis assistance.
We also thank Patricia Maina for her contribution to the preterm birth and heat
literature review and Sung Kyun Park for early stage advice.
Funding This work was supported by a Michigan Institute for Clinical and Health
Research Postdoctoral Translational Scholars Fellowship (2UL1TR000433-06),
National Institute of Environmental Health Sciences grants K99ES026198 and
P30ES017885, Cooperative Agreement Number EH001124 from the Centers for
Disease Control and Prevention (CDC), National Science Foundation grant 1520803,
and CDC/National Institute for Occupational Safety and Health grant T42 OH008455.
Its contents are solely the responsibility of the authors and do not necessarily
represent the official views of the CDC or the Michigan Department of Health and
Human Services. None of the funders participated in the design, collection, analysis
or interpretation of the data.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval The Michigan Department of Health and Human Services
Institutional Review Board (IRB) (study number 201302–03-XA) and the University
of Michigan Institutional Review Board (study number HUM00071694) determined
the study exempt from IRB review per Title 45 Code of Federal Regulations 46.101.
(b) – research involving collection of existing data and information is recorded by
the investigator in such a manner that subjects cannot be identified.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Birth outcome data were derived from birth certificate
records kept by the Michigan Department of Health and Human Services (MDHHS).
These confidential data may be obtained from the MDHHS Division for Vital Records
and Health Statistics following completion of a data use agreement and IRB
approval. The exposure data are publicly available and were obtained as described
in the Methods. The processed exposure data may be requested from C. Gronlund (
gronlund@umich.edu).
Open access This is an open access article distributed in accordance with the
Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits
others to copy, redistribute, remix, transform and build upon this work for any
purpose, provided the original work is properly cited, a link to the licence is given,
and indication of whether changes were made. See: https://creativecommons.org/
licenses/by/4.0/.
ORCID iDs
Carina J Gronlund http://orcid.org/0000-0002-0533-745X
Rachel S Bergmans http://orcid.org/0000-0001-5740-6691
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English
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Interlink between solubility, structure, surface and thermodynamics in the ThO2(s, hyd)–H2O(l) system
|
Frontiers in chemistry
| 2,022
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cc-by
| 13,486
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TYPE Original Research
PUBLISHED 15 November 2022
DOI 10.3389/fchem.2022.1042709 TYPE Original Research
PUBLISHED 15 November 2022
DOI 10.3389/fchem.2022.1042709 TYPE Original Research
PUBLISHED 15 November 2022
DOI 10.3389/fchem.2022.1042709 OPEN ACCESS OPEN ACCESS
EDITED BY
Ping Yang,
Los Alamos National Laboratory (DOE),
United States Christian Kiefer1*, Thomas Neill1,2, Nese Cevirim-Papaioannou 1,
Dieter Schild 1, Xavier Gaona1*, Tonya Vitova1, Kathy Dardenne1,
Jörg Rothe1, Marcus Altmaier1 and Horst Geckeis1 1Institute for Nuclear Waste Disposal, Karlsruhe Institute of Technology, Karlsruhe, Germany,
2Research Centre for Radwaste Disposal and Williamson Research Centre for Molecular Environmental
Science, Department of Earth and Environmental Sciences, The University of Manchester, Manchester,
United Kingdom The impact of temperature on a freshly precipitated ThO2(am, hyd) solid phase
was investigated using a combination of undersaturation solubility experiments
and a multi-method approach for the characterization of the solid phase. XRD
and EXAFS confirm that ageing of ThO2(am, hyd) at T = 80°C promotes a
significant increase of the particle size and crystallinity. TG-DTA and XPS
support that the ageing process is accompanied by an important decrease
in the number of hydration waters/hydroxide groups in the original amorphous
Th(IV) hydrous oxide. However, while clear differences between the structure of
freshly precipitated ThO2(am, hyd) and aged samples were observed, the
characterization methods used in this work are unable to resolve clear
differences between solid phases aged for different time periods or at
different pH values. Solubility experiments conducted at T = 22°C with fresh
and aged Th(IV) solid phases show a systematic decrease in the solubility of the
solid phases aged at T = 80°C. In contrast to the observations gained by solid
phase characterization, the ageing time and ageing pH significantly affect the
solubility measured at T = 22°C. These observations can be consistently
explained considering a solubility control by the outermost surface of the
ThO2(s, hyd) solid, which cannot be properly probed by any of the
techniques considered in this work. Solubility data are used to derive the
thermodynamic properties (log *K°s,0, ΔfG°m) of the investigated solid phases,
and discussed in terms of particle size using the Schindler equation. These
results provide new insights on the interlink between solubility, structure,
surface and thermodynamics in the ThO2(s, hyd)–H2O(l) system, with special
emphasis on the transformation of the amorphous hydrous/hydroxide solid
phases into the thermodynamically stable crystalline oxides. Kiefer C, Neill T,
Cevirim-Papaioannou N, Schild D,
Gaona X, Vitova T, Dardenne K, Rothe J,
Altmaier M and Geckeis H (2022),
Interlink between solubility, structure,
surface and thermodynamics in the
ThO2(s, hyd)–H2O(l) system. Front. Chem. 10:1042709. Kiefer C, Neill T,
Cevirim-Papaioannou N, Schild D,
Gaona X, Vitova T, Dardenne K, Rothe J,
Altmaier M and Geckeis H (2022),
Interlink between solubility, structure,
surface and thermodynamics in the
ThO2(s, hyd)–H2O(l) system.
Front. Chem. 10:1042709.
doi: 10.3389/fchem.2022.1042709 COPYRIGHT
© 2022 Kiefer, Neill, Cevirim-
Papaioannou, Schild, Gaona, Vitova,
Dardenne, Rothe, Altmaier and Geckeis.
This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms. thorium, solubility, surface, temperature, thermodynamics, structure Interlink between solubility,
structure, surface and
thermodynamics in the
ThO2(s, hyd)–H2O(l) system OPEN ACCESS
EDITED BY
Ping Yang,
Los Alamos National Laboratory (DOE),
United States
REVIEWED BY
Xiaofeng Guo,
Washington State University,
United States
Buzuayehu Abebe,
Adama Science and Technology
University, Ethiopia
Hongwu Xu,
Arizona State University, United States
*CORRESPONDENCE
Christian Kiefer,
kiefer@subatech.in2p3.fr
Xavier Gaona,
xavier.gaona@kit.edu
SPECIALTY SECTION
This article was submitted to Catalytic
Reactions and Chemistry,
a section of the journal
Frontiers in Chemistry
RECEIVED 12 September 2022
ACCEPTED 31 October 2022
PUBLISHED 15 November 2022
CITATION
Kiefer C, Neill T,
Cevirim-Papaioannou N, Schild D,
Gaona X, Vitova T, Dardenne K, Rothe J,
Altmaier M and Geckeis H (2022),
Interlink between solubility, structure,
surface and thermodynamics in the
ThO2(s, hyd)–H2O(l) system. Front. Chem. 10:1042709. OPEN ACCESS
EDITED BY
Ping Yang,
Los Alamos National Laboratory (DOE),
United States
REVIEWED BY
Xiaofeng Guo,
Washington State University,
United States
Buzuayehu Abebe,
Adama Science and Technology
University, Ethiopia
Hongwu Xu,
Arizona State University, United States
*CORRESPONDENCE
Christian Kiefer,
kiefer@subatech.in2p3.fr
Xavier Gaona,
xavier.gaona@kit.edu
SPECIALTY SECTION
This article was submitted to Catalytic
Reactions and Chemistry,
a section of the journal
Frontiers in Chemistry
RECEIVED 12 September 2022
ACCEPTED 31 October 2022
PUBLISHED 15 November 2022
CITATION
Kiefer C, Neill T,
Cevirim-Papaioannou N, Schild D,
Gaona X, Vitova T, Dardenne K, Rothe J,
Altmaier M and Geckeis H (2022),
Interlink between solubility, structure,
surface and thermodynamics in the
ThO2(s, hyd)–H2O(l) system. Front. Chem. 10:1042709. doi: 10.3389/fchem.2022.1042709 1 Introduction Based
on
their
own
experimental
data
and
the
reinterpretation of previous studies (Hietanen, 1954; Kraus
and Holmberg, 1954; Baes et al., 1965), the authors derived a
speciation model involving the predominance of the polynuclear
species
Th2(OH)2
6+,
Th2(OH)3
5+
and
Th6(OH)14
10+. This
chemical model was updated in the review work by Baes and
Mesmer, who reported the hydrolysis scheme and corresponding
equilibrium constants in the reference state (I = 0, T = 25°C)
including the hydrolysis species ThOH3+, Th(OH)2
2+, Th(OH)4
(aq), Th2(OH)2
6+, Th4(OH)8
8+ and Th6(OH)15
9+ (Baes and
Mesmer, 1976). In 2001, Neck and Kim published the most
comprehensive study on An (IV) solubility and hydrolysis, which
included the critical review of previously reported studies and
estimations of hydrolysis constants based on a semi-empirical
electrostatic model. For Th(IV), the authors selected a hydrolysis
scheme including the species ThOH3+, Th(OH)2
2+, Th(OH)3
+,
Th(OH)4 (aq), Th4(OH)12
4+ and Th6(OH)15
9+, and reported the
solubility product log K°s,0 (Th(OH)4, am) = –47.0 (Neck and
Kim, 2001). Neck and co-workers reported also solubility
experiments
combined
with
laser-induced
breakdown
detection (LIBD) and X-ray absorption fine structure (XAFS)
(Neck et al., 2002). The solubility product was determined to be
log K°s,0 = –47.8, whereas XAFS and LIBD confirmed the
presence of large amounts of small Th(IV) colloids within
3.5 < pH < 5. Altmaier et al. investigated the solubility and
the colloid formation of Th(IV) in 0.5 M and 5.0 M NaCl as well
as in 0.25, 2.5 and 4.5 M MgCl2 solutions (Altmaier et al., 2004). In the case of early canister failure, actinide chemistry in the
near-field of a repository for the disposal of high-level radioactive
waste (HLW) will be affected by elevated temperatures of up to
200°C, depending upon host-rock system and repository concept. The corrosion of iron occurring after the closure of the repository
and possible water access will promote reducing conditions, for
which the oxidation states + III and +IV are expected to control
the solution chemistry of the actinides, An (Grenthe et al., 2020). In aqueous systems, An (IV) behavior is dominated by the
formation
of
sparingly
soluble,
nanoparticulate
and
amorphous hydrous oxides, AnO2 (am, hyd), and by a strong
tendency for hydrolysis (Rand et al., 2009; Grenthe et al., 2020). OPEN ACCESS doi: 10.3389/fchem.2022.1042709 COPYRIGHT
© 2022 Kiefer, Neill, Cevirim-
Papaioannou, Schild, Gaona, Vitova,
Dardenne, Rothe, Altmaier and Geckeis. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms. KEYWORDS Frontiers in Chemistry frontiersin.org 01 Kiefer et al. 10.3389/fchem.2022.1042709 1 Introduction The transition of these amorphous oxy-hydroxides into the
thermodynamically stable crystalline oxides AnO2 (cr), is
kinetically
hindered
due
to their low solubility,
and is
generally not observed in aqueous systems. However, ageing
processes induced by time or temperature may facilitate this
transition with the consequent decrease of the overall solubility. Most of the available solubility studies were conducted using
amorphous hydrous oxides. The nomenclature used to define
such solid phases includes Th(OH)4(am), ThO2·xH2O(am),
ThO2(am, hyd), ThO2(am), among others, thus highlighting
the ill-defined character of the solid phase controlling the
solubility in these studies (Dzimitrowicz et al., 1985; Rai et al.,
1997; Neck and Kim, 2001; Altmaier et al., 2004; Rand et al.,
2009). Although the crystalline oxides AnO2(cr) are the
thermodynamically stable An(IV) end-members (Rand et al.,
2009; Grenthe et al., 2020), the amorphous hydrous oxides are
actually controlling An(IV) solubility in aqueous systems and
thus are considered to estimate solubility upper limits required in
the safety assessment of repositories for nuclear waste disposal. The transition of An(IV) amorphous solid/colloidal phases into
the thermodynamically stable AnO2(cr) has been correlated with
the increase of the particle-size (Neck et al., 2007). However, no
attempts have been made so far to link the loss of hydroxide
groups/water in the transformation of AnO2(am, hyd) into
AnO2(cr), or to understand how this affects the stability of
the corresponding solid phases, as well as the interlink with
surface area and particle size. q
y
The solubility and hydrolysis of thorium have been
extensively investigated in the literature, although solubility
studies
involving
a
concurrent,
thorough
solid
phase
characterization
are
sparse. Hietanen
and
co-workers
conducted potentiometric and coulometric experiments with
Th(IV) in 3 M NaCl solutions at 25°C (Hietanen and Sillen,
1968). Based
on
their
own
experimental
data
and
the
reinterpretation of previous studies (Hietanen, 1954; Kraus
and Holmberg, 1954; Baes et al., 1965), the authors derived a
speciation model involving the predominance of the polynuclear
species
Th2(OH)2
6+,
Th2(OH)3
5+
and
Th6(OH)14
10+. This
chemical model was updated in the review work by Baes and
Mesmer, who reported the hydrolysis scheme and corresponding
equilibrium constants in the reference state (I = 0, T = 25°C)
including the hydrolysis species ThOH3+, Th(OH)2
2+, Th(OH)4
(aq), Th2(OH)2
6+, Th4(OH)8
8+ and Th6(OH)15
9+ (Baes and
Mesmer, 1976). 1 Introduction The authors emphasized the relevant role of intrinsic colloids in
the aquatic chemistry of thorium in neutral and alkaline
solutions. Kobayashi et al. conducted solubility experiments
and investigated the differences on solid phases and solubility
after aging a freshly precipitated Th(IV) hydroxide at 363 K for
3–6 weeks (Kobayashi et al., 2016). The authors observed a
significant decrease on the solubility correlating with a slight
growth of the particle size in the aged solid, for which a log K°s,0 =
–51.6 was reported. Nishikawa et al. investigated the solubility of
Th(IV) in 0.5 M NaClO4 and HClO4 solutions with 2.0 < pH <
8.0 (Nishikawa et al., 2018). The starting material Th(OH)4(am)
was aged at 298 K, 313 K and 333 K for up to 40 weeks. Solubility
measurements conducted with the aged solid phases were also
conducted at 298, 313 and 333 K, and showed a systematic
decrease of solubility with increase of the ageing temperature. The expected increase of crystallinity at elevated temperatures
was confirmed by XRD measurements. In the case of early canister failure, actinide chemistry in the
near-field of a repository for the disposal of high-level radioactive
waste (HLW) will be affected by elevated temperatures of up to
200°C, depending upon host-rock system and repository concept. The corrosion of iron occurring after the closure of the repository
and possible water access will promote reducing conditions, for
which the oxidation states + III and +IV are expected to control
the solution chemistry of the actinides, An (Grenthe et al., 2020). In aqueous systems, An (IV) behavior is dominated by the
formation
of
sparingly
soluble,
nanoparticulate
and
amorphous hydrous oxides, AnO2 (am, hyd), and by a strong
tendency for hydrolysis (Rand et al., 2009; Grenthe et al., 2020). The transition of these amorphous oxy-hydroxides into the
thermodynamically stable crystalline oxides AnO2 (cr), is
kinetically
hindered
due
to their low solubility,
and is
generally not observed in aqueous systems. However, ageing
processes induced by time or temperature may facilitate this
transition with the consequent decrease of the overall solubility. The solubility and hydrolysis of thorium have been
extensively investigated in the literature, although solubility
studies
involving
a
concurrent,
thorough
solid
phase
characterization
are
sparse. Hietanen
and
co-workers
conducted potentiometric and coulometric experiments with
Th(IV) in 3 M NaCl solutions at 25°C (Hietanen and Sillen,
1968). 2.2 pH measurements pH values were measured with combination glass electrodes
(Orion ROSS), calibrated against commercial pH buffer solutions
(Merck, pH 2–10). In salt solutions of ionic strength I ≥
0.1 mol kg−1, the measured pH value (pHexp) is an operational
value related to [H+] by pHm = pHexp + Am (with [H+] in molal
units). Am is an empirical parameter accounting for the liquid
junction potential of the electrode and the activity coefficient of
H+, which is given as a function of the background electrolyte
concentration. Values of Am in NaCl solutions were taken from
the literature (Altmaier et al., 2003). 1 Introduction In 2001, Neck and Kim published the most
comprehensive study on An (IV) solubility and hydrolysis, which
included the critical review of previously reported studies and
estimations of hydrolysis constants based on a semi-empirical
electrostatic model. For Th(IV), the authors selected a hydrolysis
scheme including the species ThOH3+, Th(OH)2
2+, Th(OH)3
+,
Th(OH)4 (aq), Th4(OH)12
4+ and Th6(OH)15
9+, and reported the
solubility product log K°s,0 (Th(OH)4, am) = –47.0 (Neck and
Kim, 2001). Neck and co-workers reported also solubility
experiments
combined
with
laser-induced
breakdown
detection (LIBD) and X-ray absorption fine structure (XAFS)
(Neck et al., 2002). The solubility product was determined to be
log K°s,0 = –47.8, whereas XAFS and LIBD confirmed the
presence of large amounts of small Th(IV) colloids within
3.5 < pH < 5. Altmaier et al. investigated the solubility and
the colloid formation of Th(IV) in 0.5 M and 5.0 M NaCl as well
as in 0.25, 2.5 and 4.5 M MgCl2 solutions (Altmaier et al., 2004). The
structures
of
ThO2(am,
hyd)
nanoparticles
and
precursors, and AnO2(am, hyd) more generally, have been
extensively investigated using extended X-ray absorption fine
structure (EXAFS) analysis. Th coordination environments
found varied from aqueous Th4+-like coordination for early
hydrolysis
products,
with
Th
coordinated
by
10–13
O
backscatterers (likely H2O and OH−) at 2.44–2.51 Å and few
or no Th backscatterers (Neck et al., 2002; Rothe et al., 2002), to
more
crystalline
ThO2-like
systems
for
heat
treated
nanoparticles, with seven to nine O backscatterers at 2.41 Å
and a Th shell fit at 3.9–4.0 Å with up to 12 Th backscatterers
(Bonato et al., 2020; Manaud et al., 2020). Rothe et al. analysed
Th particles formed via hydrolysis and precipitation of colloids at
pH 1.3–3.6 (Rothe et al., 2002). The resulting EXAFS fitting for
the majority of samples indicated a coordination environment Frontiers in Chemistry frontiersin.org 02 Kiefer et al. 10.3389/fchem.2022.1042709 similar to that of aqueous Th, with only the highest pH sample
and the precipitated ‘fresh’ ThO2(am,hyd) showing evidence of
Th-Th backscatterers and long-range order. This is consistent
with the findings of Neck et al. who, using a combination of
EXAFS and laser-induced breakdown detection (LIBD), showed
the structure of Th colloids formed after titration of acidic
solution
had
high
Th-O
and
low
Th-Th
coordination
numbers relative to crystalline ThO2 (Neck et al., 2002). 1 Introduction similar to that of aqueous Th, with only the highest pH sample
and the precipitated ‘fresh’ ThO2(am,hyd) showing evidence of
Th-Th backscatterers and long-range order. This is consistent
with the findings of Neck et al. who, using a combination of
EXAFS and laser-induced breakdown detection (LIBD), showed
the structure of Th colloids formed after titration of acidic
solution
had
high
Th-O
and
low
Th-Th
coordination
numbers relative to crystalline ThO2 (Neck et al., 2002). More
recently, EXAFS studies into the structure of nanoparticulate
ThO2 and PuO2 have shown that the nearest neighbor O shell
alters significantly for very small particles (<10 nm) (Bonato
et al., 2020). This trend was quantified by the Debye-Waller
factor of the first Th-O shell, which was observed to decrease with
increasing particle size (attained by TEM) and attributed to
structural disorder which was more prevalent at smaller
nanoparticle
sizes. There
was
also
a
direct
correlation
observed
between
particle
size
and
Th-Th
coordination
number; the larger the particle size, the higher the Th-Th
coordination. This trend increased most significantly at small
particle sizes and appeared to approach full Th-Th coordination
of ThO2 with particles >25 nm in size. Other investigations into
thermally synthesized ThO2 nanoparticles treated at 220-250°C
found that the nanoparticles were highly crystalline and there
was little or no variation in particle structure at different
formation temperatures (Manaud et al., 2020). An XRD and
XAFS study into ThO2 nanoparticles of sizes from 2.5 to 33.8 nm
found that, with decreasing particle size, there was a systematic
shift to larger lattice parameters by 1.1%, which coincided with a
decrease in Th-Th coordination number, consistent with other
studies discussed here (Plakhova et al., 2019). Amidani and co-
workers investigated ThO2 nanoparticles by means of HEXS and
HERFD XANES (Amidani et al., 2021). The authors observed
mixed thorium hexamer clusters with 1 nm nanoparticles in the
initial steps of formation, which lead to more crystalline,
thermodynamically
stable
nanoparticles
when
exposed
to
elevated temperatures (150–1,000 °C). Recently, Romanchuk
et al. compiled a systematic study of AnO2 (An = Th, U, Pu)
and CeO2 nanoparticles, illustrating the similarities between the
systems and suggested that the reduced coordination numbers
observed in EXAFS fitting was a result of the core-shell nature of
AnO2 nanoparticles (Romanchuk et al., 2022). 2.1 Chemicals Thorium nitrate pentahydrate (Th(NO3)4·5H2O), sodium
chloride (NaCl), nitric acid (ultrapure), HCl Titrisol© and
NaOH Titrisol© were purchased from Merck. Ethanol (99.9%)
was obtained from VWR Chemicals. All solutions were prepared
with Milli-Q water (Milli–Q academic, Millipore, 18.2 MΩ cm). Before use, Milli-Q water was purged with Ar for >1 h to remove
traces of dissolved CO2(g). All samples were prepared and stored
in an Ar-glove box (<1 ppm O2), either at T = 22 or 80°C. 1 Introduction More
recently, EXAFS studies into the structure of nanoparticulate
ThO2 and PuO2 have shown that the nearest neighbor O shell
alters significantly for very small particles (<10 nm) (Bonato
et al., 2020). This trend was quantified by the Debye-Waller
factor of the first Th-O shell, which was observed to decrease with
increasing particle size (attained by TEM) and attributed to
structural disorder which was more prevalent at smaller
nanoparticle
sizes. There
was
also
a
direct
correlation
observed
between
particle
size
and
Th-Th
coordination
number; the larger the particle size, the higher the Th-Th
coordination. This trend increased most significantly at small
particle sizes and appeared to approach full Th-Th coordination
of ThO2 with particles >25 nm in size. Other investigations into
thermally synthesized ThO2 nanoparticles treated at 220-250°C
found that the nanoparticles were highly crystalline and there
was little or no variation in particle structure at different
formation temperatures (Manaud et al., 2020). An XRD and
XAFS study into ThO2 nanoparticles of sizes from 2.5 to 33.8 nm
found that, with decreasing particle size, there was a systematic
shift to larger lattice parameters by 1.1%, which coincided with a
decrease in Th-Th coordination number, consistent with other
studies discussed here (Plakhova et al., 2019). Amidani and co-
workers investigated ThO2 nanoparticles by means of HEXS and
HERFD XANES (Amidani et al., 2021). The authors observed
mixed thorium hexamer clusters with 1 nm nanoparticles in the
initial steps of formation, which lead to more crystalline,
thermodynamically
stable
nanoparticles
when
exposed
to
elevated temperatures (150–1,000 °C). Recently, Romanchuk
et al. compiled a systematic study of AnO2 (An = Th, U, Pu)
and CeO2 nanoparticles, illustrating the similarities between the
systems and suggested that the reduced coordination numbers
observed in EXAFS fitting was a result of the core-shell nature of
AnO2 nanoparticles (Romanchuk et al., 2022). context of high-level waste disposal. At a more fundamental level,
the work provides new insights on the structural evolution of the
ThO2(s, hyd) solid phases in the transition from amorphous to
crystalline, with special focus on the role of water and surface
effects. context of high-level waste disposal. At a more fundamental level,
the work provides new insights on the structural evolution of the
ThO2(s, hyd) solid phases in the transition from amorphous to
crystalline, with special focus on the role of water and surface
effects. 2.3 Solid phase preparation and solubility
experiments with ThO2(s, hyd) Molar concentrations of elemental species are
calculated by atomic concentrations and curve fit results. Data
analysis was performed using ULVAC-PHI MultiPak program,
version 9.9. pHm of the 0.1 M NaOH suspensions was significantly lower at
T = (80 ± 2) °C, i.e. pHm (T = 80°C) = 11.2. microscope (now Thermo Fisher Scientific Inc.) was used to
image the carbon coated sample surfaces. The primary electron
beam energy was 30 keV. Relative atomic concentrations (H not
detected) were calculated by areas of elemental lines of survey
spectra, recorded at 187.85 eV pass energy of the analyzer, after
subtraction of a local Shirley background and taking into account
sensitivity factors and asymmetry parameters of elemental lines,
and the transmission function of the analyzer (Moulder et al., 1995;
Briggs and Grant, 2003). Relative error of semiquantitative atomic
concentrations is typically within ± (10–20)%. Curve fits to narrow
scans of elemental lines recorded at 23.5 eV pass energy were
performed by Gaussian functions after Shirley background
subtraction. Molar concentrations of elemental species are
calculated by atomic concentrations and curve fit results. Data
analysis was performed using ULVAC-PHI MultiPak program,
version 9.9. pHm of the 0.1 M NaOH suspensions was significantly lower at
T = (80 ± 2) °C, i.e. pHm (T = 80°C) = 11.2. Different series of undersaturation solubility experiments at
T = 22°C were prepared using the fresh and aged solid phases
described above. Each independent batch sample was prepared
with 1.5–3 ml of the corresponding solid suspension (fresh
precipitate or solid phases aged at T = 80°C). This aliquot was
centrifuged for 5 min at 4,000 g, separated from the supernatant,
and washed two times with the corresponding equilibration
solution. After the last washing step, the solid phase was
contacted
with
5–20 ml
(depending
upon
pHm)
of
the
equilibration solution. Seven series of solubility experiments
were prepared using a freshly precipitated ThO2(am, hyd),
and ThO2(s, hyd) aged for 1, 2 and 4.5 months (pHm = 3 and
12.8) at T = (80 ± 2)°C. These solid phases were equilibrated at
T = 22°C in 0.1 M HCl–NaCl solutions with 2.3 < pHm < 6.3. Concentration of Th and pH were monitored at regular time
intervals until equilibrium conditions were attained (defined by
constant [Th] and pHm readings). 2.3 Solid phase preparation and solubility
experiments with ThO2(s, hyd) For the determination of
thorium concentrations, an aliquot of the supernatant of each
sample was centrifuged (12,000 g) with 10 kDa filters (NanoSep
Merck Millipore, pore size ~2 nm). A given volume of the
resulting filtrate was diluted with 2% ultrapure HNO3, and Th
concentration was quantified by ICP-MS (Perkin Elmer ELAN
6100). Thermogravimetric analysis (TG) with differential thermal
analysis (DTA) were performed under Ar atmosphere using a
Netzsch STA 449C equipment. Measurements were performed
with 10–20 mg of dry solid material. Samples were heated up to
1,200 °C with a heating rate of 10 K min−1. 2.4.2 EXAFS measurements Th L3 edge XAFS spectra were recorded at the INE beamline
of the KIT Light Source (KARA storage ring), Karlsruhe,
Germany (Rothe et al., 2019). Ge (4 2 2) crystals were used in
the
Lemonnier-type
double
crystal
monochromator. The
monochromated
radiation
was
focused
by
a
Rh-coated
toroidal mirror resulting in a spot size of <1 mm at the
sample position. EXAFS measurements were performed with
selected solid samples, i.e. ThO2(am, hyd, fresh) and three aged
solid phases ThO2(s, hyd, aged): t = 1 m at pHm = 3, t = 1 m at
pHm = 12.8 and t = 5.5 m at pHm = 12.8. Approximately
10–15 mg of each solid phase was washed once with a weakly
alkaline solution (pHm ≈9.2) and re-suspended in a small
volume of the same solution. The resulting suspensions were
placed into a sealed, liquid nitrogen stable sample holder. The
plastic cells were contained under Ar atmosphere in a gas-tight
cell and transported to the synchrotron source, where they were
stored under Ar atmosphere until the EXAFS measurements. Samples were measured at 80 K in a liquid nitrogen cooled
cryostat
in
fluorescence
acquisition
mode
simultaneously
using a 1-pixel and a 4-pixel silicon drift (Vortex) detector. The excitation energy scale was calibrated using a ThO2
reference
standard
(first
inflection
point
calibrated
to
16,300 eV). The resulting XAFS spectra were processed and
analysed
using
Athena
and
Artemis
software
from
the
Demeter software package (FEFF 6) (Ravel and Newville,
2005). The spectra were fit using a ‘shell-by-shell’ approach
using fixed coordination numbers to limit number of fit
parameters, and the statistical validity of each shell was
verified by way of an F-test (>95% validity) (Downward et al.,
2007). 2.3 Solid phase preparation and solubility
experiments with ThO2(s, hyd) A232Th(IV) stock solution was prepared by slow titration of a
0.15 M Th(NO3)4 solution to pH ≈10–11 using 1.0 M NaOH. The resulting solid, i.e. ThO2(am, hyd), was separated from the
nitrate-rich
supernatant
by
centrifugation
at
4,000 g
for
10–15 min. The solid phase was dissolved in 0.1 M HCl, and
the procedure was repeated until nitrate was washed out
(<10 ppm, determined with colorimetric test strips Merck
MQuant®). Approximately 1.2 g of ThO2(am, hyd) solid phase
was obtained in a final slow titration, which was divided into nine
aliquots of approximately 130 mg each. Eight of the aliquots were
contacted with either 0.1 M HCl–NaCl (pHm ≈3) or 0.1 M
NaOH (pHm = 12.8) at T = (22 ± 2) °C, and aged at T =
(80 ± 2)°C for 1, 2, 4.5 and 5.5 months. The remaining aliquot was
used for the study of the freshly precipitated hydrous oxide. Due
to the temperature-dependence of the pKw of water, the actual In this context, this work aims at investigating the impact of
temperature, ageing time and pH on the structure and solubility
of a freshly precipitated ThO2(am, hyd) solid phase. A multi-
method approach including XRD, SEM, XPS, TG-DTA and
EXAFS is used to thoroughly characterize the resulting solid
phases, with special focus on particle size, degree of hydration
and surface properties. In combination with solubility data, this
information is used to derive thermodynamic properties for the
solid
phases
investigated,
which
are
compared
with
thermodynamic data selected in the NEA-TDB reference
database. The results contribute to the quantitative description
of radionuclide aqueous systems of potential relevance in the Frontiers in Chemistry frontiersin.org 03 Kiefer et al. 10.3389/fchem.2022.1042709 microscope (now Thermo Fisher Scientific Inc.) was used to
image the carbon coated sample surfaces. The primary electron
beam energy was 30 keV. Relative atomic concentrations (H not
detected) were calculated by areas of elemental lines of survey
spectra, recorded at 187.85 eV pass energy of the analyzer, after
subtraction of a local Shirley background and taking into account
sensitivity factors and asymmetry parameters of elemental lines,
and the transmission function of the analyzer (Moulder et al., 1995;
Briggs and Grant, 2003). Relative error of semiquantitative atomic
concentrations is typically within ± (10–20)%. Curve fits to narrow
scans of elemental lines recorded at 23.5 eV pass energy were
performed by Gaussian functions after Shirley background
subtraction. 2.4 Solid phase characterization 2.4.1 XRD, TG-DTA, SEM and XPS 2.4.1 XRD, TG-DTA, SEM and XPS X-ray powder diffraction (XRD) measurements were performed
with a Bruker AXS D8 Advance X-Ray powder diffractometer (Cu-
Kα radiation, LynxEye XE-T detector). An aliquot with approximately
1–2 mg of each solid phase was washed 3 times with 0.5 ml of ethanol
to remove the matrix solutions. After the last washing step, the solid
phase was re-suspended in ethanol and deposited as suspension on a
spot prepared with vaseline on a XRD sample plate. The
measurement angle was 2° < 2θ < 70° with incremental steps of
0.015° and a measurement time of 0.4 s for each step. Diffractograms
collected were compared with reference data reported in the Joint
Committee on Powder Diffraction Standard (JCPDS, 2001). Based on
the full width at half maximum (FWHM) of the diffraction peaks, the
crystallite size for a given solid was calculated using the Scherrer
equation (Scherrer, 1918; Holzwarth and Gibson, 2011). An aliquot of the washed solid was prepared on an indium foil,
dried under Ar atmosphere, and subsequently analyzed by X-ray
photoelectron
spectroscopy
(XPS)
and
scanning
electron
microscopy (SEM). XPS measurements were performed with a
XPS system PHI 5000 VersaProbe II (ULVAC-PHI Inc.) equipped
with a scanning microprobe X-ray source (monochromatic Al Kα,
hν = 1,486.7 eV). Binding energies of elemental lines are charge
referenced to the oxidic portion of the O 1s spectrum at 530.0 eV. A FEI Quanta 650 FEG environmental scanning electron Frontiers in Chemistry frontiersin.org 04 Kiefer et al. 10.3389/fchem.2022.1042709 FIGURE 1
Diffractograms of the Th(IV) solid phases synthesized in this
work and equilibrated at T = 80°C and pHm(25°C) = 3 and 12.8,
except for the sample “ThO2(freshly precipitated)”, which was
measured 2 days after precipitation. Vertical dashed lines
refer to the ThO2(cr) reference (PDF 75-0052). The low quality of
the diffractogramm obtained for the sample aged for 4.5 months
at pHm = 12.8 was caused by the limited amount of solid phase
available. The diffractogram obtained for the freshly precipitated material
shows a main broad feature at 2Θ ≈28.5°, which reflects the
amorphous character of the solid phase. This is in line with
previous observations reported by Kobayashi and co-workers for
freshly precipitated Th(IV) hydrous oxide (Kobayashi et al., 2016). In
all other cases, XRD patterns show narrower peaks with well-defined
2Θ positions, reflecting a higher degree of crystallinity for the Th(IV)
aged samples. 2.4.1 XRD, TG-DTA, SEM and XPS No significant differences are visually observed in the
diffractograms of Th(IV) solid phases aged for different times or at
different pHm values. The peak positions in the XRD of the aged
samples are in excellent agreement with reference data reported for
ThO2(cr) in the JCPDS database, with main reflections at 2Θ = 27.6,
32.0, 45.9, 54.4 and 57.0°. A good agreement is also obtained with the
peak positions in the XRD reported by Kobayashi et al. for Th(IV)
solid phases aged at T = 90°C for 3–6 weeks in 0.1–2.0 M NaClO4 and
0.1–3.0 M NaCl (Kobayashi et al., 2016). Plakhova and co-workers
reported well-resolved XRD patterns for freshly precipitated ThO2(s,
hyd) (Plakhova et al., 2019). However, we note that the drying process
at T = 40 and 150°C followed by the authors may have increased the
degree of crystallinity of the originally precipitated Th(IV) solid phase. Table 1 shows the results of the Scherrer analysis based on the
evaluation of the fullwidthat half maximum(FWHM) intensityof the
XRD peaks in Figure 1. Large differences are observed between the
crystallite size of freshly precipitated and aged ThO2(s, hyd) solid
phases, whereas very similar crystallite sizes are quantified for solid
phases aged at T = 80°C for samples aged for different lengths of time. For the same ageing time, the Scherrer analysis hints towards the
formation of slightly larger crystallites in the solid phases aged at
pHm = 3 than for those aged at pHm = 12.8, although the values of the
crystallite size overlap within their uncertainties. This observation
could be rationalized by the higher solubility of Th(IV) at pHm = 3
(≈10−2 M, calculated at T = 25°C for ThO2(am, hyd, aged)) than at
pHm = 12.8 (≈10−8 M), which may result in a faster particle growth
through enhanced dissolution and precipitation reactions. Kobayashi et al. reported crystallite sizes between 3.1 and 4 nm for
selected Th(IV) solid phases aged at T = 90°C (Kobayashi et al., 2016), in
moderate agreement with crystallite sizes determined in this work. Plakhova and co-workers reported crystallite sizes of ≈2 and ≈3.6 nm
forThO2(s,hyd)solidphasesprecipitatedatroomtemperature(butdried
at T = 40 and 150°C) in 3.0 M NH3·H2O and 3.0 M NaOH, respectively
(Plakhova et al., 2019). 1
The most correct definition of the solid refers to ThOx(OH)y·zH2O(s),
with 2x + y = 4. FIGURE 1 FIGURE 1 Diffractograms of the Th(IV) solid phases synthesized in this
work and equilibrated at T = 80°C and pHm(25°C) = 3 and 12.8,
except for the sample “ThO2(freshly precipitated)”, which was
measured 2 days after precipitation. Vertical dashed lines
refer to the ThO2(cr) reference (PDF 75-0052). The low quality of
the diffractogramm obtained for the sample aged for 4.5 months
at pHm = 12.8 was caused by the limited amount of solid phase
available. Diffractograms of the Th(IV) solid phases synthesized in this
work and equilibrated at T = 80°C and pHm(25°C) = 3 and 12.8,
except for the sample “ThO2(freshly precipitated)”, which was
measured 2 days after precipitation. Vertical dashed lines
refer to the ThO2(cr) reference (PDF 75-0052). The low quality of
the diffractogramm obtained for the sample aged for 4.5 months
at pHm = 12.8 was caused by the limited amount of solid phase
available. 3 Results and discussion
3.1 Solid phase characterization In the process of particle growth through ageing (either at
room or elevated temperatures), a decrease of the number of
hydration waters in the solid ThO2(am, hyd)1 is expected. The
number of hydration waters in An (IV) hydrous oxides has been 2.4.1 XRD, TG-DTA, SEM and XPS The hydrothermal treatment of Th(IV) solid
phases in H2O (T = 210°C, pH not specified) and 3.0 M NaOH (T =
180°C) resulted in particle sizes of ≈4.3 and ≈4.7 nm, respectively. 3.1.1 XRD Figure 1 shows the powder diffractograms collected for the
fresh and aged Th(IV) solid phases investigated within this study. The figure includes also the main diffraction lines reported for
the ThO2(cr) reference (PDF 75-0052). Frontiers in Chemistry frontiersin.org 05 Kiefer et al. 10.3389/fchem.2022.1042709 the ThO2(s, hyd, fresh) and ThO2(s, hyd, aged) solid phases investigated in this work. n.d. stands for not determined. y
2( ,
y ,
)
2( ,
y ,
g
)
p
g
Sample
Crystallite size [nm]
ThO2(s, hyd, fresh, T = 22°C)
(1.5 ± 0.5)
ThO2(s, hyd, aged, t = 1 m, pHm = 3, T = 80°C)
(4.4 ± 0.5)
ThO2(s, hyd, aged, t = 1 m, pHm = 12.8, T = 80°C)
(4.1 ± 0.5)
ThO2(s, hyd, aged, t = 2 m, pHm = 3, T = 80°C)
(4.7 ± 0.5)
ThO2(s, hyd, aged, t = 2 m, pHm = 12.8, T = 80°C)
(4.1 ± 0.5)
ThO2(s, hyd, aged, t = 4.5 m, pHm = 3, T = 80°C)
(4.8 ± 0.5)
ThO2(s, hyd, aged, t = 4.5 m, pHm = 12.8, T = 80°C)
n.d
ThO2(s, hyd, aged, t = 5.5 m, pHm = 3, T = 80°C)
(4.6 ± 0.5)
ThO2(s, hyd, aged, t = 5.5 m, pHm = 12.8, T = 80°C)
(4.1 ± 0.5) Sample ThO2(s, hyd, fresh, T = 22°C)
ThO2(s, hyd, aged, t = 1 m, pHm = 3, T = 80°C)
ThO2(s, hyd, aged, t = 1 m, pHm = 12.8, T = 80°C)
ThO2(s, hyd, aged, t = 2 m, pHm = 3, T = 80°C)
ThO2(s, hyd, aged, t = 2 m, pHm = 12.8, T = 80°C)
ThO2(s, hyd, aged, t = 4.5 m, pHm = 3, T = 80°C)
ThO2(s, hyd, aged, t = 4.5 m, pHm = 12.8, T = 80°C)
ThO2(s, hyd, aged, t = 5.5 m, pHm = 3, T = 80°C)
ThO2(s, hyd, aged, t = 5.5 m, pHm = 12.8, T = 80°C) ThO2(s, hyd, fresh, T = 22°C)
ThO2(s, hyd, aged, t = 1 m, pHm = 3, T = 80°C)
ThO2(s, hyd, aged, t = 1 m, pHm = 12.8, T = 80°C)
ThO2(s, hyd, aged, t = 2 m, pHm = 3, T = 80°C)
ThO2(s, hyd, aged, t = 2 m, pHm = 12.8, T = 80°C)
ThO2(s, hyd, aged, t = 4.5 m, pHm = 3, T = 80°C)
ThO2(s, hyd, aged, t = 4.5 m, pHm = 12.8, T = 80°C)
ThO2(s, hyd, aged, t = 5.5 m, pHm = 3, T = 80°C)
ThO2(s, hyd, aged, t = 5.5 m, pHm = 12.8, T = 80°C) TABLE 2 Weight loss and calculated number of hydration waters as quantified by TG-DTA for the Th(IV) hydrous phases investigated in this work. Sample
Weight loss
Hydration water
ThO2(s, hyd, fresh, T = 22°C)
(16.7 ± 0.5) %
(2.9 ± 0.1)
ThO2(s, hyd, aged, t = 1 m, pHm = 3, T = 80°C)
(9.1 ± 0.5) %
(1.5 ± 0.1)
ThO2(s, hyd, aged, t = 1 m, pHm = 12.8, T = 80°C)
(7.7 ± 0.5) %
(1.2 ± 0.1)
ThO2(s, hyd, aged, t = 2 m, pHm = 3, T = 80°C)
(7.6 ± 0.5) %
(1.2 ± 0.1)
ThO2(s, hyd, aged, t = 2 m, pHm = 12.8, T = 80°C)
(8.8 ± 0.5) %
(1.4 ± 0.1)
ThO2(s, hyd, aged, t = 4.5 m, pHm = 3, T = 80°C)
(9.9 ± 0.5) %
(1.6 ± 0.1)
ThO2(s, hyd, aged, t = 5.5 m, pHm = 12.8, T = 80°C)
(8.0 ± 0.5) %
(1.3 ± 0.1) calculated number of hydration waters as quantified by TG-DTA for the Th(IV) hydrous phases investigated in this work. Sample Solids aged at T =
80°C: (2) 1 month at pHm = 3; (3) 1 month at pHm = 12.8; (4)
2 months at pHm = 3; (5) 2 months at pHm = 12.8; (6)
4.5 months at pHm = 3; (7) 4.5 months at pHm = 12.8; (8)
5.5 months at pHm = 3; (9) 5.5 months at pHm = 12.8. Both bulk (XRD, TG-DTA) and surface-sensitive techniques
(XPS) considered for the characterization of the solid phases
show no clear effect of ageing time or pH on the properties of the
investigated Th(IV) hydrous oxides. However, it is important to
bear in mind that XPS provides an information depth of about
3 nm at the experimental conditions. Alterations affecting a
monolayer at the Th(IV) oxide surface will most likely not be
clearly identified by this technique. the number of hydration waters in M(IV)O2 (s, hyd) as a function
of time, with M = Th (this work), U (Cevirim-Papaioannou,
2018) and Tc (Yalcintas et al., 2016; Grenthe et al., 2020). The
figure shows a clear qualitative trend to decrease the number of
hydration waters with ageing time, thus supporting the
transformation
of
hydroxide/hydrated
phases
into
the
corresponding, thermodynamically stable, oxides. Sample often assumed as 2 (i.e. AnO2·2H2O(s) An(OH)4(s)), although
a few previous studies report clearly lower values of hydration
waters (0.6–1) for M(IV) solid phases aged for 1–3 years at room
temperature (Yalcintas et al., 2016; Cevirim-Papaioannou, 2018). TG-DTA measurements were performed to determine the
number of hydration waters of freshly precipitated ThO2(am,
hyd) as well as of solid phases aged at T = 80°C for t = 1, 2, 4.5 and
5.5 months. The main quantitative outcome evaluated from the
TG-DTA data is the total weight loss measured up to 1,200°C,
which is assigned to the number of hydration waters in the
investigated hydrous oxides (see Table 2). This evaluation
approach is a simplification of the actual situation, where
loosely bound water, sticking moisture, hydroxide groups and
(less likely) crystal waters might be present. The results in the
table clearly show a significantly larger amount of hydration
waters in the freshly precipitated solid (n = 2.9 ± 0.1) compared
to the aged phases (n = 1.4 ± 0.2). No clear trend is observed for
the latter as a function of time or pH. The values of hydration
water determined for the aged solid phases are in line with data
reported by Cevirim-Papaioannou for a U(IV) hydrous oxide
phase aged at T = 22°C for up to 798 days (n = 1.0 ± 0.5)
(Cevirim-Papaioannou, 2018). Figure 2 shows the evolution of FIGURE 2
Evolution of the number of hydration waters in M(IV)O2 (s,
hyd) with time as reported in this work for Th or reported in the
literature for U (Cevirim-Papaioannou, 2018) and Tc (Yalcintas
et al., 2016; Grenthe et al., 2020). Figure modified after
(Grenthe et al., 2020) (Tc chapter, prepared by B. Grambow). FIGURE 2
Evolution of the number of hydration waters in M(IV)O2 (s,
hyd) with time as reported in this work for Th or reported in the
literature for U (Cevirim-Papaioannou, 2018) and Tc (Yalcintas
et al., 2016; Grenthe et al., 2020). Figure modified after
(Grenthe et al., 2020) (Tc chapter, prepared by B. Grambow). Evolution of the number of hydration waters in M(IV)O2 (s,
hyd) with time as reported in this work for Th or reported in the
literature for U (Cevirim-Papaioannou, 2018) and Tc (Yalcintas
et al., 2016; Grenthe et al., 2020). Figure modified after
(Grenthe et al., 2020) (Tc chapter, prepared by B. Grambow). Sample Frontiers in Chemistry 06 frontiersin.org 10.3389/fchem.2022.1042709 Kiefer et al. 2 months at pHm = 3, as well as the narrow scans of the Th 4f and
the O 1s lines. The narrow scan of the O 1s line includes also the
fit with the hydrate, hydroxide and oxide contributions. The XP
spectra of all investigated solids are shown together in Figures
3A,B. Table 3 summarizes the atom % of Th and O as well as the
ratio [O]/[Th] quantified considering the intensities of the Th 4f
and the O 1s lines. In all cases, the ratio O: Th is well above 2
(2.3–2.8),
thus
underpinning
the
significant
presence
of
hydroxide and hydrate groups that enhance the ratio [O]/[Th]
beyond the value of two present in the crystalline ThO2(cr). Table 3 summarizes also the speciation of oxygen (as hydrate,
hydroxide or oxide) resulting from the fit of the O 1s line. FIGURE 3
XPS narrow scans of Th(IV) hydrous oxides: (A) Th 4f lines; (B)
O 1s lines. (1) freshly precipitated solid phase. Solids aged at T =
80°C: (2) 1 month at pHm = 3; (3) 1 month at pHm = 12.8; (4)
2 months at pHm = 3; (5) 2 months at pHm = 12.8; (6)
4.5 months at pHm = 3; (7) 4.5 months at pHm = 12.8; (8)
5.5 months at pHm = 3; (9) 5.5 months at pHm = 12.8. Consistently
with
other
solid
phase
characterization
techniques considered in this work (XRD, TG-DTA), XPS
provides clear evidence of the differences between the freshly
precipitated Th(IV) hydrous oxide and the corresponding solids
aged at T = 80°C. However, no clear trends are observed in the
XPS analysis of solid phases aged for different times or at
different pH values. The fraction of oxide calculated as
average of all solid phases equilibrated at pHm = 3 (58.8 ±
5.8%) is virtually the same as the average obtained for solid
phases equilibrated at pHm = 12.8 (58.6 ± 4.9%). However, the
oxide content in all aged samples is clearly above the fraction of
oxide quantified for the freshly precipitated Th(IV) hydrous
oxide (43.3%). FIGURE 3
XPS narrow scans of Th(IV) hydrous oxides: (A) Th 4f lines; (B)
O 1s lines. (1) freshly precipitated solid phase. 3.1.3 EXAFS Solid phase
Atom % Th
Atom % O
Ratio [O]/[Th]
O2- (%)
OH−(%)
H2O (%)
ThO2(s, hyd, fresh, T = 22°C)
27.6
72.4
2.6
43.3
47.9
8.8
ThO2(s, hyd, aged, t = 1 m, pHm = 3, T = 80°C)
30.4
69.6
2.3
56.1
30.5
13.4
ThO2(s, hyd, aged, t = 1 m, pHm = 12.8, T = 80°C)
28.7
71.3
2.5
63.5
29.8
6.7
ThO2(s, hyd, aged, t = 2 m, pHm = 3, T = 80°C)
29.7
70.3
2.4
59.3
31.6
9.1
ThO2(s, hyd, aged, t = 2 m, pHm = 12.8, T = 80°C)
26.1
73.9
2.8
57.0
33.8
9.2
ThO2(s, hyd, aged, t = 4.5 m, pHm = 3, T = 80°C)
28.0
72.0
2.6
55.6
30.8
13.6
ThO2(s, hyd, aged, t = 4.5 m, pHm = 12.8, T = 80°C)
29.3
70.7
2.4
55.6
26.9
17.5
ThO2(s, hyd, aged, t = 5.5 m, pHm = 3, T = 80°C)
29.0
71.0
2.5
64.0
28.9
7.1
ThO2(s, hyd, aged, t = 5.5 m, pHm = 12.8, T = 80°C)
28.6
71.4
2.5
58.4
31.6
10.0 Atom % Th
Atom % O
Ratio [O]/[Th]
O2- (%)
OH−(%)
H2O (%) 3.1.3 EXAFS 10.3389/fchem.2022.1042709 TABLE 3 Atomic % of Th and O, ratio [O]/[Th] and speciation of oxygen (as atomic percent of oxide, hydroxide and water) as calculated from the Th 4f
and O 1s lines in the XPS measurements. Relative error of atomic concentrations is within ± (10–20) %. ThO
Th
fidi
i t
t
ith
i
EXAFS fitti
th
th
th
l
lik l d
t
h
ll
t llit
TABLE 3 Atomic % of Th and O, ratio [O]/[Th] and speciation of oxygen (as atomic percent of oxide, hydroxide and water) as calculated from the Th 4f
and O 1s lines in the XPS measurements. Relative error of atomic concentrations is within ± (10–20) %. Solid phase
Atom % Th
Atom % O
Ratio [O]/[Th]
O2- (%)
OH−(%)
H2O (%)
ThO2(s, hyd, fresh, T = 22°C)
27.6
72.4
2.6
43.3
47.9
8.8
ThO2(s, hyd, aged, t = 1 m, pHm = 3, T = 80°C)
30.4
69.6
2.3
56.1
30.5
13.4
ThO2(s, hyd, aged, t = 1 m, pHm = 12.8, T = 80°C)
28.7
71.3
2.5
63.5
29.8
6.7
ThO2(s, hyd, aged, t = 2 m, pHm = 3, T = 80°C)
29.7
70.3
2.4
59.3
31.6
9.1
ThO2(s, hyd, aged, t = 2 m, pHm = 12.8, T = 80°C)
26.1
73.9
2.8
57.0
33.8
9.2
ThO2(s, hyd, aged, t = 4.5 m, pHm = 3, T = 80°C)
28.0
72.0
2.6
55.6
30.8
13.6
ThO2(s, hyd, aged, t = 4.5 m, pHm = 12.8, T = 80°C)
29.3
70.7
2.4
55.6
26.9
17.5
ThO2(s, hyd, aged, t = 5.5 m, pHm = 3, T = 80°C)
29.0
71.0
2.5
64.0
28.9
7.1
ThO2(s, hyd, aged, t = 5.5 m, pHm = 12.8, T = 80°C)
28.6
71.4
2.5
58.4
31.6
10.0
FIGURE 4
EXAFS (left) and Fourier-Transformed EXAFS (right) for (from top to bottom) ThO2(s, hyd) freshly precipitated, ThO2(s, hyd) aged, t = 1 m, pHm =
3, ThO2(s, hyd) aged, t = 1 m, pHm = 12.8 and ThO2(s, hyd) aged, t = 5.5 m, pHm = 12.8. Black lines are experimental data and red lines are fits. 3.1.3 EXAFS Th L3 edge EXAFS data and fits are shown in Figure 4, and
details of best fits are presented in Table 4. Freshly precipitated as
well as solid phases aged at T = 80 °C for 1 month (pHm = 3 and
12.8) and 5.5 months (pH = 12.8) were analysed. All samples
were fit with one or two O shells at 2.37–2.54 Å and a Th
backscatterer at (3.96 ± 0.04) Å, with all three aged samples
also including a distant O shell at (4.58 ± 0.04) Å. These distances
are in general agreement with the structure of crystalline ThO2
(Wyckoff, 1963), however there are some deviations from the
crystalline structure which are more prominent in the less aged
samples. For the fresh and 1 month aged samples, at both pHm
3 and 12.8, best fits contain a split first shell coordination. Here,
two O shells at (2.37 ± 0.03) and (2.53 ± 0.04) Å were fit,
compared to one O shell at 2.43 Å that would be expected for
crystalline ThO2. The two shells have coordination numbers
between 4.8 and three each, with total coordination ranging from
7.8 to 8.7 for each fit, which is in close agreement with the
coordination number of eight for the single O shell in crystalline All SEM pictures show irregular aggregates of ≈20 to
≈100 nm (see Figure SI-1 in Supporting Information). No
clear trend can be observed as function of the ageing time or
ageing pH, which is likely related to the very small and similar
particle size observed for all aged samples. Similar aggregates
were previously reported for other An(IV)O2(am, hyd) systems
(with M = Tc, U, Np, Pu) precipitated at room temperature
(Fellhauer, 2013; Yalcintas, 2015; Cevirim-Papaioannou, 2018). Note that much more regular aggregates were obtained in recent
studies investigating the size and local environment of Th(IV)
nanoparticles
exposed
to
much
higher
temperatures
(400–1,000°C) (Amidani et al., 2019; Bonato et al., 2020). Supplementary
Figures
SI-2A–C
in
the
Supporting
Information
exemplarily
shows
the
complete
survey
XP
spectrum of the Th(IV) sample equilibrated at T = 80°C for Supplementary
Figures
SI-2A–C
in
the
Supporting
Information
exemplarily
shows
the
complete
survey
XP
spectrum of the Th(IV) sample equilibrated at T = 80°C for Frontiers in Chemistry frontiersin.org 07 Kiefer et al. Solid phase FIGURE 4
EXAFS (left) and Fourier-Transformed EXAFS (right) for (from top to bottom) ThO2(s, hyd) freshly precipitated, ThO2(s, hyd) aged, t = 1 m, pHm =
3, ThO2(s, hyd) aged, t = 1 m, pHm = 12.8 and ThO2(s, hyd) aged, t = 5.5 m, pHm = 12.8. Black lines are experimental data and red lines are fits. FIGURE 4 FIGURE 4
EXAFS (left) and Fourier-Transformed EXAFS (right) for (from top to bottom) ThO2(s, hyd) freshly precipitated, ThO2(s, hyd) aged, t = 1 m, pHm =
3, ThO2(s, hyd) aged, t = 1 m, pHm = 12.8 and ThO2(s, hyd) aged, t = 5.5 m, pHm = 12.8. Black lines are experimental data and red lines are fits. the other three samples, likely due to a much smaller crystallite
size and/or much higher structural disorder resulting in reduced
long-range coordination, seen as a reduced Th backscatterer
coordination number. ThO2. These findings are consistent with previous EXAFS fitting
of ThO2(am,hyd) nanoparticles (Neck et al., 2002), and this is
representative of a distortion of the crystal structure in the fresh
and 1 month aged precipitates. However, this distortion does not
appear to affect the longer distance backscatterers for all fits, with
Th and O backscatterers fit at distances anticipated for a ThO2-
like, fluorite-type structure. The main difference between the fits
is in the fresh sample, where the best fit includes only 5.5 Th
backscatterers, as opposed to 12 Th backscatterers anticipated for
crystalline ThO2. This value is much closer for the sample aged
1 month at pH = 3 (11.6), 1 month at pH = 12.8 (12) and
5.5 months at pH = 12.8 (11.4) fits. These results are consistent
with the fresh sample having significantly different structure to Overall, EXAFS fitting suggests that the structure of these
nanoparticles becomes more crystalline over time. The fresh
sample has the lowest Th backscatterer coordination number
and a split first O shell, the two 1 month aged samples retain this
split O shell, but have Th backscatterer coordination numbers
more closely matching ThO2 and also a distant O shell at (4.58 ±
0.04) Å. Finally, the 5.5 months aged sample has a single O shell
with a Th-O distance identical to that of crystalline ThO2, as well
as Th and distant O backscatterers present with coordination Frontiers in Chemistry frontiersin.org 08 Kiefer et al. Solid phase 10.3389/fchem.2022.1042709 TABLE 4 EXAFS fit data for select solid samples. Solid phase
Path
N
R (Å)
σ2
ΔE0
R
ThO2(s, hyd, fresh, T = 22°C)
Th-O1
4.8
2.38 (2)
0.004 (3)
4.8 (10)
0.015
Th-O2
3.0
2.54 (3)
0.004 (3)
4.8 (10)
Th-Th
5.5
3.95 (3)
0.015 (5)
4.8 (10)
ThO2(s, hyd, aged, t = 1 m, pHm = 3, T = 80°C)
Th-O1
4.8
2.37 (3)
0.003 (1)
6.7 (15)
0.020
Th-O2
3.4
2.53 (4)
0.003 (1)
6.7 (15)
Th-Th
11.6
3.96 (1)
0.005 (3)
6.7 (15)
Th-O3
15.0
4.58 (4)
0.008 (1)
6.7 (15)
ThO2(s, hyd, aged, t = 1 m, pHm = 12.8, T = 80°C)
Th-O1
4.7
2.37 (2)
0.004 (2)
8.1 (11)
0.016
Th-O2
4.0
2.53 (2)
0.006 (3)
8.1 (11)
Th-Th
12.0
3.97 (1)
0.005 (1)
8.1 (11)
Th-O3
20.0
4.58 (2)
0.004 (0)
8.1 (11)
ThO2(s, hyd, aged, t = 4.5 m, pHm = 12.8, T = 80°C)
Th-O1
6.8
2.43 (1)
0.006 (2)
9.9 (10)
0.020
Th-Th
11.4
3.96 (1)
0.005 (1)
9.9 (10)
Th-O2
17.3
4.56 (2)
0.007 (1)
9.9 (10)
ThO2(cr) (Rothe et al., 2002)
Th-O1
8.0
2.41 (1)
0.0054
2.3
0.0241
Th-Th
12
3.98 (2)
0.0042
4.7
Th-O2
24
4.63 (1)
0.0064
6.4
Coordination numbers (N), U bond distances (R (Å)), Debye-Waller factors (σ2), shift in energy from calculated Fermi level (ΔE0) and ‘goodness of fit’ factor (R). Coordination numbers
were fixed. Numbers in parentheses are the standard deviation on the last decimal place. Coordination numbers (N), U bond distances (R (Å)), Debye-Waller factors (σ2), shift in energy from calculated Fermi level (ΔE0) and ‘goodness of fit’ factor (R). Coordination numbers
were fixed. Numbers in parentheses are the standard deviation on the last decimal place. numbers similar to those in the crystalline structure (Table 1, see
data reported by Rothe et al., 2002). with experimental data reported by Kobayashi and co-workers
for a solid phase aged at T = 90°C during 6–8 weeks. Note
however that Kobayashi et al. followed a different ageing
approach as used in this study–each independent solubility
sample was aged at T = 90°C at the target pH (ranging
between ≈1.5 and ≈9), whereas in the present work the solid
phase used in each solubility series was aged at a single pH. Solid phase Because of the impact of pH in the ageing process (see next
paragraph and Figure 5), the different ageing approach followed
in Kobayashi et al. and in this work may lead to differences in the
solubility data, especially in the less acidic samples. Recently,
Nisbet and co-workers investigated the solubility of ThO2(cr) in
the temperature range 150–250°C (Nisbet et al., 2018). The
authors did not report solubility constants for the investigated
systems, but the measured Th concentrations are clearly lower as
those observed in the present study, in line with the crystalline
character of their solid phase. 3.2 Solubility experiments Figures 5A–D shows solubility of Th(IV) determined in this
work for freshly precipitated ThO2(am, hyd, fresh) and ThO2(s,
hyd, aged) aged at T = 80 °C for t = 1, 2, 4.5 m and pHm = 3, 12.8. The figure also shows the solubility curves corresponding to
freshly precipitated, aged and crystalline ThO2 solid phases
calculated
using
the
solubility
and
hydrolysis
constants
selected in the NEA-TDB (Rand et al., 2009), as well as
solubility
curves
calculated
with
the
solubility
constants
derived in this work (see Section 3.3). The experimental data on the freshly precipitated Th(IV)
hydrous oxide agrees well with the solubility calculated for
ThO2(am, hyd, fresh) using the NEA-TDB thermodynamic
selection (Figure 5A). Experimental data are also in moderate
agreement with previous studies reporting the solubility of
ThO2(am, hyd) at T = 25°C in 0.1 M NaCl or NaClO4 (Ryan
and Rai, 1987; Rai et al., 2000). All aged Th(IV) solid phases show
lower solubilities than ThO2(am, hyd, fresh), consistent with the
increase in particle size/crystallinity observed by XRD. This
observation supports also that aged solids are not a mixture of
(nano-)crystalline and amorphous phases, as in this case the
solubility should be defined by the most soluble solid, i.e. the
amorphous phase. Solubility data obtained for the Th(IV) solid
phase aged for 2 months at pHm = 3 is also in good agreement Experimental data in Figure 5 show that the solubility of the
Th(IV) hydrous oxide slightly decreases with the ageing time at
T = 80°C. Unexpectedly, the pH in which the Th(IV) solid phase
was aged has a very significant effect on the solubility measured at
T = 22°C. Hence, the solid phases aged at pHm = 3 show up to two
orders of magnitude lower solubility than the solid phases aged at
pHm = 12.8. This effect is reproduced for the solid phases aged
during 1, 2 and 4.5 months. These observations are apparently in
contradiction with the minor differences observed by XRD, TG-
DTA and XPS for Th(IV) solid phases aged for different contact
times and at different pH values. However, these results can be
rationalized by a solubility control established by a few Frontiers in Chemistry frontiersin.org 09 Kiefer et al. 3.2 Solubility experiments 10.3389/fchem.2022.1042709 FIGURE 5
Experimental solubility data obtained in this work and model calculations using thermodynamic data derived in this work or reported in the
NEA-TDB for Th(IV) hydrous oxide: (A) freshly precipitated; (B) aged for 1m at T = 80°C and pHm = 3 and 12.8; (C) aged for 2m at T = 80°C and pHm =
3 and 12.8; (D) aged for 4.5m at T = 80°C and pHm = 3 and 12.8. FIGURE 5
Experimental solubility data obtained in this work and model calculations using thermodynamic data derived in this work or reported in the
NEA-TDB for Th(IV) hydrous oxide: (A) freshly precipitated; (B) aged for 1m at T = 80°C and pHm = 3 and 12.8; (C) aged for 2m at T = 80°C and pHm =
3 and 12.8; (D) aged for 4.5m at T = 80°C and pHm = 3 and 12.8. surfacial ThOx (OH)y (H2O)z species finally leading to a reduced
solubility. Moreover, differences in the surface charge of the
ThO2(s, hyd) (positive at pHm = 3, negative at pHm = 12.8) can
possibly affect to the ageing process, and consequently influence
solubility. monolayers of the ThO2(s, hyd) surface. Such few monolayers
have a minor weight in bulk characterization methods (XRD,
TG-DTA) but also in “surface-sensitive” methods like XPS,
which
provides
average
values
of
a
≈3 nm
layer. This
hypothesis is also in line with previous studies by Grambow,
Vandenborre and co-workers (Vandenborre et al., 2010;
Grambow
et
al.,
2017),
who
claimed
that
solubility
measurements of ZrO2(s), ThO2(s) and UO2(s) are not
representative of the bulk phase, but are rather controlled by
surface processes of a few monolayers of the corresponding
oxide. Although the starting materials used in these studies
were crystalline oxides sintered at very high temperatures
(400–1,000°C), the authors claimed that “solubility” of Th(IV)
system was controlled by “ThOx (OH)y (H2O)z” present in the
grain boundaries. Solid phase The values of hydration water x = 2.9 and x =
1.4 have been considered for the modelling of the freshly
precipitated and aged solid phases, respectively, where x =
(1.4 ± 0.3) is the average value of the number of hydration
waters quantified for all aged solid phases. The impact of x in the
current modelling calculations is however very minor in the
conditions of this study, because the water activity in 0.1 M NaCl
is close to unity, i.e. aw = 0.9966. The outcome of this modelling
exercise is shown in Figure 5, whereas Table 5 summarizes the log
*K°s,0 values determined for all investigated Th(IV) hydrous
oxides as compared to the values selected by NEA-TDB. where the values of *β°(n,m) are know from the NEA-TDB and γH
+
is calculated using the SIT formalism (Ciavatta, 1980; Grenthe
et al., 2020). The values of hydration water x = 2.9 and x =
1.4 have been considered for the modelling of the freshly
precipitated and aged solid phases, respectively, where x =
(1.4 ± 0.3) is the average value of the number of hydration
waters quantified for all aged solid phases. The impact of x in the
current modelling calculations is however very minor in the
conditions of this study, because the water activity in 0.1 M NaCl
is close to unity, i.e. aw = 0.9966. The outcome of this modelling
exercise is shown in Figure 5, whereas Table 5 summarizes the log
*K°s,0 values determined for all investigated Th(IV) hydrous
oxides as compared to the values selected by NEA-TDB. Differences in the solubility of ThO2(am, hyd) solid phases have
been often attributed to the effect of particle size (and by the extension
of the surface area) in the Gibbs energy of formation (ΔfG°m) of the
solid (Neck et al., 2007; Kobayashi et al., 2016). The Schindler equation
correlates the ΔfG°m of an amorphous/colloidal solid phase with the
ΔfG°m of the corresponding crystalline phase considering a term with
the surface contribution (Schindler, 1967; Neck et al., 2007): The solubility constant determined in this work for
ThO2(am, hyd, fresh) (log K°s,0 = 46.7 ± 0.4) is in excellent
agreement with the value selected in the NEA-TDB, log K°s,0 =
(46.7 ± 0.9). 3.3 Thermodynamic evaluation of
solubility phenomena Experimental solubility data were modelled with the aim of
determining
the
solubility
constants
(log
*K°s,0)
of
the
investigated Th(IV) hydrous oxides. The model of the system
controlling the solubility of Th(IV) includes both monomeric
(Th4+, ThOH3+, Th(OH)2
2+, Th(OH)4 (aq)) and polyatomic
(Th2(OH)2
6+,
Th2(OH)3
5+,
Th4(OH)8
8+,
Th4(OH)12
4+,
Th6(OH)15
9+, Th6(OH)14
10+) aqueous species as selected in the
NEA-TDB (Rand et al., 2009), as well as the solid phases
ThO2(am, hyd, fresh) and ThO2(ncr, hyd, t, pHm), with t = 1,
2, 4.5months and pHm = 3, 12.8. The term “ncr” indicates the
nanocrystalline character of the Th(IV) hydrous oxides solid
phases obtained after the hydrothermal ageing at T = 80°C. The As already discussed above, the in general higher solubility of
Th(IV) at lower pH induces faster recrystallization rates,
concomitant with somewhat larger diameters of crystalline
units. Even though the characterization methods applied in
the present work are not able to resolve variations in the first
surface monolayers, we assume that the higher recrystallization
rate at low pH may also have an impact on the structure of Frontiers in Chemistry frontiersin.org 10 Kiefer et al. 10.3389/fchem.2022.1042709 TABLE 5 Solubility constants for ThO2(s, hyd) and ThO2(cr) as determined in this work or selected in the NEA-TDB (Rand et al., 2009). The values of log
*K°s,0 and log K°s,0 correspond to the equilibrium reactions ThO2·nH2O(s) + 4 H+ 5 Th4+ + (2 + n) H2O(l) and ThO2·nH2O(s) + (2–n) H2O(l) 5 Th4+ +
4 OH−
ti
l TABLE 5 Solubility constants for ThO2(s, hyd) and ThO2(cr) as determined in this work or selected in the NEA-TDB (Rand et al., 2009). The values of log
*K°s,0 and log K°s,0 correspond to the equilibrium reactions ThO2·nH2O(s) + 4 H+ 5 Th4+ + (2 + n) H2O(l) and ThO2·nH2O(s) + (2–n) H2O(l) 5 Th4+ +
4 OH−, respectively. 3.3 Thermodynamic evaluation of
solubility phenomena Solid phase
log *K°s,0
log K°s,0
Source
ThO2(am, hyd, fresh, T = 22°C)
(9.3 ± 0.4)
–(46.7 ± 0.4)
present work
ThO2(am, hyd, fresh)
(9.3 ± 0.9)
–(46.7 ± 0.9)
NEA-TDB
ThO2(am, hyd, aged)
(8.5 ± 0.9)
–(47.5 ± 0.9)
NEA-TDB
ThO2(ncr, hyd, t = 1 m, pHm = 3, T = 80°C)
(5.8 ± 0.2)
–(50.2 ± 0.2)
present work
ThO2(ncr, hyd, t = 2 m, pHm = 3, T = 80°C)
(6.2 ± 0.4)
–(49.8 ± 0.4)
present work
ThO2(ncr, hyd, t = 4.5 m, pHm = 3, T = 80°C)
(5.2 ± 0.5)
–(50.8 ± 0.5)
present work
ThO2(ncr, hyd, t = 1 m, pHm = 12.8, T = 80°C)
(7.8 ± 0.5)
–(48.2 ± 0.5)
present work
ThO2(ncr, hyd, t = 2 m, pHm = 12.8, T = 80°C)
(7.6 ± 0.1)
–(48.4 ± 0.1)
present work
ThO2(ncr, hyd, t = 4.5 m, pHm = 12.8, T = 80°C)
(6.8 ± 0.4)
–(49.2 ± 0.4)
present work
ThO2(crystalline)
(1.76 ± 1.11)
–(54.24 ± 1.11)
NEA-TDB Solid phase aging period. However, the most significant effect on the values of
log K°s,0 is observed as a function of the ageing pH. Hence, in
average, the samples aged at pHm = 3 have a solubility constant of
ca. 2 log10-units lower than the solid phases aged at pHm = 12.8. This behavior is a strong evidence of the effect of pH on the
recrystallization rate, which is higher at pHm = 3 due to the
higher solubility of Th(IV) under acidic conditions. modelling approach is based on the fit of log *Ks,0°, whereas the
values of log *β(n,m)° are kept constant as selected by NEA-TDB. modelling approach is based on the fit of log *Ks,0°, whereas the
values of log *β(n,m)° are kept constant as selected by NEA-TDB. The datasets collected for each solid phase are fitted individually
by minimizing the function ((log[Th]exp–log[Th]calc)2)1/2. The
value [Th]calc is the sum of the species [Th4+] [ThOH3+]
[Th(OH)2
2+]
[Th(OH)4
(aq)]
[Th2(OH)2
6+]
[Th2(OH)3
5+]
[Th4(OH)8
8+]
[Th4(OH)12
4+]
[Th6(OH)15
9+]
and
[Th6(OH)14
10+], and can be calculated as: The datasets collected for each solid phase are fitted individually
by minimizing the function ((log[Th]exp–log[Th]calc)2)1/2. The
value [Th]calc is the sum of the species [Th4+] [ThOH3+]
[Th(OH)2
2+]
[Th(OH)4
(aq)]
[Th2(OH)2
6+]
[Th2(OH)3
5+]
[Th4(OH)8
8+]
[Th4(OH)12
4+]
[Th6(OH)15
9+]
and
[Th6(OH)14
10+], and can be calculated as: The strong impact of the ageing time and pH on the value of
log K°s,0 is however not reflected in differences observed by the
different solid phase characterization techniques considered in
this work, i.e. XRD, TG-DTA, SEM, XPS and XAFS. As discussed
in Section 3.2, this likely reflects that solubility phenomena in the
investigated system is mostly controlled by surface processes
occurring
in
the
first
monolayer
of
the
Th(IV)
oxide
(0.2–0.4 nm). Indirect
evidences
pointing
to
the
same
hypothesis were reported by Vandenborre and co-workers on
the
basis
of
isotopic
exchange
experiments
with
229Th
(Vandenborre et al., 2010; Grambow et al., 2017). [Th]calc *K°
s,0 γH+ 4 mH+ 4 aw
−(2+x)
+ *β°
(n.m)
*K°
s,0
n γH+ 4n−m mH+ 4n−m aw
m−n(2+x)
(1) (1) where the values of *β°(n,m) are know from the NEA-TDB and γH
+
is calculated using the SIT formalism (Ciavatta, 1980; Grenthe
et al., 2020). 4 Summary and conclusion In order to avoid the bias introduced by the contribution of
ΔfG°m (H2O, l) in Th(IV) oxide phases with different number of
hydration waters, the following reaction is considered for the
calculation of the Gibbs energy of formation of the solid: The influence of temperature on Th(IV) solid phases and
solubility
was
systematically
investigated
under
Ar
atmosphere using a freshly precipitated ThO2(am, hyd)
solid phase as starting material. The fresh precipitate was
aged at T = 80°C for different time periods (t = 1, 2, 4.5, 5.5 m)
and at two different pHm values (pHm = 3.0, 12.8). The
resulting solid phases were comprehensively characterized
using a multimethod approach including XRD, TG-DTA,
SEM, XPS and XAFS techniques. Selected solid phases were
used for the preparation of solubility experiments at T = 22°C
in 0.1 M NaCl solutions with 2.3 ≤pHm ≤7.0. These data, in
combination
with
the
hydrolysis
constants
and
SIT
coefficients selected in the NEA-TDB, were used to derive
the corresponding solubility constants (log K°s,0) and assess
the systematic variations caused by the ageing at elevated
temperatures. ThO2(s) + 4 H+ # Th4+ + 2 H2O(l)
(7) (7) which leads to: ΔfG°
m(ThO2) RT ln *K°
s,0 + ΔfG°
mTh4+ + 2 ΔfG°
m(H2O)
−4 ΔfG°
m(H+)
(8) (8) where *K°
s,0 are the solubility constants calculated using the
model derived in this study. The values of ΔfG°m for Th4+,
H2O(l) and H+ were taken from the Th book of the NEA-
TDB (Rand et al., 2009). The comparison of experimental
data with predictions using the Schindler equation is shown
in Figure 6. Figure 6 shows qualitatively a good agreement between ΔfG°m
calculated
from
experimental
solubility
data
and
model
predictions using the Schindler equation. A somehow large
deviation is observed for the freshly precipitated Th(IV)
hydrous oxide, whereas ΔfG°m determined for the aged phases
agree well with model calculations. Experimental evidences
obtained in this work provide an indirect link between
solubility phenomena in the ThO2(s) system with surface
properties of a (few) monolayer(s) of the solid. Although
surface is unequivocally playing a key role in the solubility The ageing of a freshly precipitated ThO2(am, hyd) solid
phase at T = 80°C induces a significant increase of the
crystallinity
and
particle
size,
as
confirmed
by
XRD
measurements. Solid phase This supports the validity of the solubility
experiments and modelling approach, and represents a sound
anchoring point for the quantification of the solubility constants
of the aged phases investigated in this work. A very significant
decrease in the solubility constant (≥1.5 log10-units) with respect
to the freshly precipitated solid phase is observed for the aged
solid phases already after 1 month of ageing time. A further
decrease in the values of log Ks,0° is observed with the increase of (2) ΔfG°m(ThO2(amcol)) ΔfG°
m(ThO2(cr)) + 23γS
( where ΔfG°m is directly related to the solubility constant through
the Gibbs energy of reaction (ΔrG°m), S is the molar surface that
depends on the particle size, and γ is the mean free surface energy
per unit surface area of solid-liquid interface. The molar surface
is defined as S Mα
ρd where M is the molecular weight, ρ is the
density of the solid, d is the particle size and α is a geometrical
shape factor, α ≈6 for spherical particles (Neck et al., 2007). According to Schindler, γ can be estimated as (Schindler, 1967): Frontiers in Chemistry frontiersin.org 11 Kiefer et al. 10.3389/fchem.2022.1042709 γ −
3RTlnK°
sp(S →0)
2NA4πri2
(3) FIGURE 6
Application of Schindler equation to the ThO2(s) system and
comparison with experimental data determined in the present
work. Figure modified from (Neck et al., 2007). (3) (4) The Schindler equation acknowledges the key role of the
surface on the overall stability of a solid phase, and thus on
its
solubility
constant. Although
the
assumption
of
homogeneous spherical particles is not confirmed in the
present
work,
experimental
solubility
constants
determined in this study in combination with particle size
determined by the Scherrer equation have been compared
with model predictions using the Schindler equation. The
molar standard Gibbs energy values for ThO2 solid phases of
the present work are calculated according to the following
equations: equilibrium, it is less evident that particle size is the driving
force controlling solubility in the ThO2(s) as assumed in the
Schindler equation. ln K°
sp −ΔRGm(T)
RT
(5)
ΔRGm(T) ΔfGm(Products) − ΔfGm(Educts)
(6) (5) 4 Summary and conclusion TG-DTA and XPS support that this process
is accompanied by an important decrease in the number of
hydration waters/hydroxide groups in the original amorphous
Th(IV) hydrous oxide, ThOx (OH)y·zH2O(s). EXAFS data
show a decreased number of Th-Th backscatterers at ≈ Frontiers in Chemistry frontiersin.org 12 Kiefer et al. 10.3389/fchem.2022.1042709 Publisher’s note All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher. Author contributions XG, MA, and HG defined the initial concept of the study. XG,
NC, and CK contributed to conception and design of the
experimental work. CK and TN performed the experiments
and conducted data evaluation. DS conducted the SEM and
XPS measurements, and corresponding data evaluation. TV,
KD, and JR revised the EXAFS data evaluation. CK, XG, and
TN wrote the first draft of the manuscript. All authors
contributed to manuscript revision, read, and approved the
submitted version. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Acknowledgments 3.96 Å in the freshly precipitated solid phase, however in the
solid phases aged at T = 80°C the number of Th-Th
backscatterers
approaches
the
ThO2(cr)
bulk
value. Nevertheless, all solid characterization methods used in this
work are unable to resolve clear differences between solid
phases aged for different time periods or at different pHm
values. Frank Geyer, Annika Kaufmann, Stephanie Kraft and Melanie
Böttle (all KIT–INE) are gratefully acknowledged for the ICP–MS/
OES, TG-DTA measurements and technical support. This work was
partially funded by the German Federal Ministry for Education and
Research under contract 02NUK039A, the ThermAc project. The
Institute
for
Beam
Physics
and
Technology
(KIT-IBPT)
is
acknowledged
for
the
operation
of
the
Karlsruhe
Research
Accelerator (KARA), and provision of beamtime at the INE-
Beamline operated by the Institute for Nuclear Waste Disposal at
the KIT Light Source. TN and TV acknowledge funding from the
EuropeanResearch Council(ERC) ConsolidatorGrant2020 underthe
European Union’s Horizon 2020 research and innovation programme
(grant agreement no. 101003292). We acknowledge support by the
KIT-Publication Fund of the Karlsruhe Institute of Technology. Solubility experiments with fresh and aged Th(IV) solid
phases conducted at T = 22°C show a clear decrease in the
solubility of the solid phases aged at T = 80°C. In contrast to the
observations gained by solid phase characterization, the ageing
time and (specially) ageing pHm are shown to have a very
important impact on the solubility measured at T = 22°C. These
apparently discordant observations can be explained in a
consistent manner by claiming a solubility control by a few
monolayers
in
the
surface
of
the
ThO2(s,
hyd)
solid
(0.2–0.4 nm), which cannot be properly probed by any of the
bulk (XRD, TG-DTA, SEM, XAFS) or surface-sensitive (XPS,
with a penetration depth of ca. 4 nm) techniques considered in
this work. Data availability statement The raw data supporting the conclusion of this article will be
made available by the authors, without undue reservation. Supplementary material The Supplementary Material for this article can be found
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determine the solubility product and EXAFS for aqueous speciation. Radiochim. Acta 90, 485–494. doi:10.1524/ract.2002.90.9-11_2002.485 Yalcintas, E. (2015). Redox, solubility and sorption chemistry of technetium in
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Institute of Technology. Frontiers in Chemistry frontiersin.org 14
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SOIL MESO- AND MACROFAUNA IN TWO SOYBEAN CROPS AFTER SWINE WASTEWATER APPLICATION
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Engenharia agrícola
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KEYWORDS: mineral fertilization, swine wastewater, soil fauna. KEYWORDS: mineral fertilization, swine wastewater, soil fauna. _________________________
1 Grupo de Pesquisa em Ciências Agro-Ambientais, Universidade Estadual do Oeste do Paraná/ Cascavel - PR, Brasil.
Received in: 8-8-2016
Accepted in: 10-10-2016
Eng. Agríc., Jaboticabal, v.37, n.3, p.556-564, ma 2*Corresponding author. Grupo de Pesquisa em Ciências Agro-Ambientais, Universidade Estadual do Oeste do Paraná/
Cascavel - PR, Brasil. E-mail: silvio.sampaio@unioeste.br 2*Corresponding author. Grupo de Pesquisa em Ciências Agro-Ambientais, Universidade Estadual do Oeste do Paraná/
Cascavel - PR, Brasil. E-mail: silvio.sampaio@unioeste.br ABSTRACT: Hog raising generates a large amount of residues that is commonly discarded into the
soil as fertilizer even with environmental risks. In this context, the aim of this study was to assess
the application effects of different doses of swine wastewater (SW) associated with mineral
fertilization on the abundance and diversity of organisms of soil meso- and macrofauna in two
soybean crops. Treatments consisted of four doses (0, 100, 200, and 300 m3 ha−1) of SW with or
without mineral fertilization in two soybean crop, totaling 24 experimental units. Soil meso- and
macrofauna were sampled using pitfall traps installed at each plot. Samples were sorted and the
organisms were separated and identified. The highest abundances were found between groups of
springtails and spider mites. Soil meso- and macrofauna differed between soybean crops influenced
by soil physical and chemical parameters. Doses of swine wastewater between 0 and 300 m3 ha−1
and mineral fertilization do not have effects on ecological indices of soil meso- and macrofauna. However, periodic applications over time change soil physicochemical variables, which may lead to
negative effects in the long term. Journal of the Brazilian Association of Agricultural
Engineering
ISSN: 1809-4430 (on-line) Journal of the Brazilian Association of Agricultural
Engineering
ISSN: 1809-4430 (on-line) INTRODUCTION Population growth stimulated livestock expansion due to increased demand for animal
protein-based food. However, intensive animal farming generates a considerable amount of residues
with high levels of contaminants, which may cause several environmental impacts. These problems
are related to a high load of organic matter, micro, and macronutrients (BASSO et al., 2012; FIA et
al., 2015). Due to inadequate management, these residues are often released or disposed of in the
environment, mainly contaminating water resources and soil. Swine wastewater reuse as a biofertilizer is an option to dispose of and/or treat the effluent by
supplying plants with water and nutrients, reducing the use of natural resources (KESSLER et al.,
2013). However, the use of wastewater in large doses may lead to the accumulation of residues in
soils (LUCAS et al., 2013), percolated water, leading to the leaching of ions (PRIOR et al., 2009;
MAGGI et al., 2011), and in runoff material (WANG et al., 2013), contaminating water resources. Thus, researches on residue physical and chemical properties are essential for planning actions that
allow recovering contaminated areas or preserving those non-degraded (DOS REIS et al., 2013). Soil chemical changes are directly influenced by microbiological activity and soil meso- and
macrofauna, which can be changed by the addition of organic effluents as a function of food supply,
temperature modification, and soil cover (MOURA et al., 2016; TESSARO et al., 2016). The
effects on soil fauna can be positive or negative, varying according to residue composition and
management, which are capable of altering the quality and quantity of soil organic matter, the C/N
ratio, and metal accumulation such as copper and zinc present in high amounts in swine residues
(POSTMA-BLAAUW et al., 2012). Therefore, the knowledge of functional groups soil meso- and
macrofauna from areas used for swine wastewater management can provide information on the
impact generated in the soil from the elimination of one or more soil organisms (BLOUIN et al.,
2013). Eng. Agríc., Jaboticabal, v.37, n.3, p.556-564, maio/jun. 2017 Soil meso- and macrofauna in two soybean crops after swine wastewater application 557 Hog raising residues may bring risks to the environment when improperly managed such as
damage to soil fauna. However, soil fauna behavior due to organic effluent reuse is still little
known. Although studies such as those developed by TESSARO et al. (2011), TESSARO et al. (2013), and PORTILHO et al. INTRODUCTION (2011) are pioneers and provide valuable answers, they were carried
out during only one crop cycle. Thus, the aim of this study was to assess the application effects of
swine wastewater (SW) doses associated with mineral fertilization (MF) on the community
structure of organisms present in soil meso- and macrofauna in two soybean crops. MATERIAL AND METHODS The experiment was carried out from October 2010 to March 2011 (crop 1) and from
November 2011 to March 2012 (crop 2), the periods indicated for soybean cultivation. The
experimental design used was a 4 × 2 factorial randomized block design with three replications,
totaling 24 sample units. Randomization followed the order established by PRIOR et al. (2009),
maintaining the application rate history of SW and MF in the area. Treatments with the respective
factor levels are shown in Table 1. Unit samples consisted of drainage lysimeters with an area of 1.0
m2, a depth of 1.1 m, and a spacing of 0.5 m. TABLE 1. Description of treatments and chemical characterization of swine wastewater. MF
SW doses (m3 ha−1)
0
100
200
300
Absence (A)
SW0–MFA
SW100–MFA
SW200–MFA
SW300–MFA
Presence (P)
SW0–MFP
SW100–MFP
SW200–MFP
SW300–MFP
SW chemical characteristics
Parameter (mg L−1)
Soybean crop season
2010–2011
2011–2012
Total organic carbon1
684.80
2916.30
Total nitrogen2
481.70
707.00
Total phosphorus3
22.06
33.01
Potassium4
8.95
265.00
Sodium4
36.70
16.80
Calcium4
52.87
236.00
Magnesium4
67.70
67.00
Copper4
1.86
8.30
Zinc4
10.22
39.00
Iron4
8.95
27.40
Manganese4
2.68
7.10
Total solids5
3498.30
20000.00
Fixed solids5
1310.80
2300.00
Volatile solids5
2187.50
17700.00
SW: Swine wastewater; MF: Mineral fertilization; 1TOC; 2Micro-Kjeldahl; 3Visible spectrophotometer; 4Flame atomic absorption
spectrophotometer; 5Gravimetric method. ABLE 1. Description of treatments and chemical characterization of swine wastewater. SW: Swine wastewater; MF: Mineral fertilization; 1TOC; 2Micro-Kjeldahl; 3Visible spectrophotometer; 4Flame atomic absorption
spectrophotometer; 5Gravimetric method. SW: Swine wastewater; MF: Mineral fertilization; 1TOC; 2Micro-Kjeldahl; 3Visible spectrophotometer; 4Flame atomic absorption
spectrophotometer; 5Gravimetric method. The soil of each lysimeter was collected before SW application and after soybean harvest for The soil of each lysimeter was collected before SW application and after soybean harvest for Eng. Agríc., Jaboticabal, v.37, n.3, p.556-564, maio/jun. 2017 Ana P. C. Maciel, Silvio C. Sampaio, Marcelo B. Remor, et al. 558 both crops for physicochemical characterization. SW used in the experiment was collected from an
integrated biosystem of swine residue treatment in the district of Três Bocas, Toledo, PR, Brazil. SW was applied in doses pre-established by PRIOR (2009) throughout the lysimeter area. MATERIAL AND METHODS 2017 559 Soil meso- and macrofauna in two soybean crops after swine wastewater application summarized in a single Principal Component Analysis (PCA) by using the software PC-ORD 4.0
(McCUNE & MEFFORD, 1999). This analysis reduces the set of original variables in a set of
Principal Components (PCs), which seeks to maintain the maximum variability of the original set. PCA was performed on the Pearson correlation matrix of variables and the criterion for retaining
PCs adopted was the broken-stick, i.e. with eigenvalues higher than expected at random
(JACKSON, 1993). In order to interpret the meaning of retained PCs of original variables, only
Pearson correlation coefficients higher than 70% were considered (JOLLIFFE, 1986). In addition,
PCA graph was constructed only with the centroids of each treatment to reduce visual pollution and
facilitate visualization. PCs validated by the broken-stick criterion were submitted to the two-factorial permutation-
based multivariate analysis of variance (Per-MANOVA) using 9999 permutations and the
Euclidean distance measure at 5% significance level to verify a significant statistical difference
between collection points and between hydrological stations. Per-MANOVA is a non-parametric
correspondent of MANOVA with the difference that the p-value is calculated by permutation of
results between treatments (ANDERSON, 2001). Per-MANOVA was performed in an 81 × 21
scheme with the first factor as treatments (Table 1) and the second factor as soybean crops (2010–
2011 and 2011–2012). The canonical correspondence analysis (CCA) (TER BRAAK, 1986) was used to infer on the
influences of soil physicochemical variables in soil meso- and macrofauna community. In the case
of structuring and correlation of taxa to the environment, both significantly higher than those
expected at random by the Monte Carlo test with 9998 randomizations (p<0.05), CCA ordering was
performed and the influence of each soil physical and chemical variable was inferred by means of
the canonical correlation coefficient. In order to reduce visual pollution and facilitate visualization,
CCA graph was constructed only with the centroids of each treatment. MATERIAL AND METHODS SW was
collected the day before the treatment application and was characterized chemically considering the
following parameters: pH, electrical conductivity (EC), total solids (TS), total fixed solids (TFS),
total volatile solids (TVS), total nitrogen (N), nitrate + nitrite (NO3− + NO2−), ammoniacal nitrogen,
total phosphorus (P), potassium (K+), sodium (Na+), calcium (Ca+2), magnesium (Mg+2), copper
(Cu+2), zinc (Zn+2), BOD, and COD. Soybean sowing was carried out two days after SW application under the no-tillage system at
each lysimeter, with spacing between lines and plants as recommended for this crop. MF was
performed in the furrow sowing with mineral fertilizer at a dose of 250 kg ha−1 using the fertilizer
formulation 0–20–20, providing 25 kg ha−1 of P2O5 and K2O for the crop. Cultural treatments were
performed at all treatments using the recommended products and dosages when pests attacked the
crop. Soil meso- and macrofauna were sampled using pitfall traps installed individually at each
lysimeter. Two collections were carried out during each soybean crops at the early (V2) and
intermediate (R2) soybean growth stages. Traps were activated for seven days at each collection
period. After this period, traps were removed and washed for the total preservative solution (4%
formalin) removal. Each container’s contents were poured into a 100-μm mesh sieve. The
specimens were stored in bottles containing 70% alcohol solution for later identification. Identification process was carried out at the level of taxonomic order using a stereoscopic
microscope and dichotomous classification keys (GALLO et al., 2002; AQUINO et al., 2006). Weather data of temperature, humidity, and precipitation during the collection periods were
provided by the Paraná State Meteorological System (SIMEPAR), as shown in Figure 1. FIGURE 1. Data of total precipitation and averages of humidity and air temperature in the soybean
crop seasons of 2010–2011 and 2011–2012. FIGURE 1. Data of total precipitation and averages of humidity and air temperature in the soybean
crop seasons of 2010–2011 and 2011–2012. Ecological indices (number of taxa, number of individuals, dominance, Simpson’s diversity,
Shannon’s diversity, Buzas and Gibson’s uniformity, Brillouin index, Menhinick’s richness,
Margalef’s richness, equitability, Fisher’s alpha diversity, Berger-Parker dominance, and Chao1
richness) of soil meso- and macrofauna related to each treatment was calculated by means of the
software PAST 3.07 (HAMMER et al., 2001). The set of ecological variables of treatments was Eng. Agríc., Jaboticabal, v.37, n.3, p.556-564, maio/jun. RESULTS AND DISCUSSION In the first soybean crop (2010–2011), 11,543 organisms were found at the soybean growth
stage V2 and 7,761 organisms at the stage R2, totaling 19,304 organisms. In the second soybean
crop (2011–2012), 23,094 organisms were found at the soybean growth stage V2 and 3,166
organisms at the stage R2, totaling 26,264. During both crops, 45,564 organisms of soil meso- and
macrofauna were collected distributed among the orders Collembola, Hymenoptera, Hemiptera,
Coleoptera, Diptera, Orthoptera, Araneae, Thysanoptera, Diplopoda, Oribatida, Mesostigmata,
Prostigmata, and Astigmata. The number of individuals collected over this study was much higher
than that collected in the study conducted by CASTALDELLI et al. (2015), who collected 15,424
organisms, demonstrating the robustness and representativeness of sampling performed in this
study. PCA of ecological indices of each treatment is shown in Figure 2. Two PCs were considered
as adequate to be assessed according to the broken-stick criterion, totaling 86.04% of the data set
variability. PCA demonstrated that the variability of ecological indices between soybean crops
(2010–2011 and 2011–2012) is higher than the variability between treatments (Figure 2). The
greater variability of ecological indices between crops is due to weather conditions before sowing
and during cultivation and collection periods. According to ALMEIDA et al. (2013), some groups
of soil meso- and macrofauna present higher incidence during wet periods. In addition, water is a
limiting factor for soil fauna behavior, presenting a direct relationship with temperature,
precipitation, and humidity. However, some groups may be negatively influenced by high humidity
due to changes in the availability of resources (CASTALDELLI et al., 2015). Another contributing factor is the change in soil physical and chemical characteristics caused
by periodic SW application (TESSARO et al., 2016). Between both soybean crops, two SW
applications were carried out in the sample area for corn and black oat cultivation, respectively. In
addition, the area has a six-year SW application history and in general three applications per year. Eng. Agríc., Jaboticabal, v.37, n.3, p.556-564, maio/jun. 2017 Ana P. C. Maciel, Silvio C. Sampaio, Marcelo B. Remor, et al. Ana P. C. Maciel, Silvio C. Sampaio, Marcelo B. Remor, et al. 560 FIGURE 2. Principal component analysis of ecological indices obtained at each treatment. FIGURE 2. Principal component analysis of ecological indices obtained at each treatment. RESULTS AND DISCUSSION Based on PC2, the 2011–2012 crop season presented
the highest number of individuals collected. The two-factorial Per-MANOVA (Table 2) with a significance of 5% confirms PCA (Figure
2), showing that the ecological indices presented statistical differences only between crops (p-value
= 0.0082). Therefore, SW doses applied (0, 100, 200, and 300 m3 ha−1) do not have significant
effects on ecological indices of soil meso- and macrofauna. SEGAT et al. (2015) did not find toxic
effects of SW on bioindicators in soils with high clay and organic matter concentrations since these
soils have a higher negative charge, which increases the cation adsorption, making the metals
unavailable for soil solution. Because this experiment was conducted in a clayey Oxisol
(GONCALVES et al., 2012; SANTOS et al., 2015), higher doses than those studied are required
(>300 m3 ha−1) so that toxic compounds (heavy metals) are available to influence soil meso- and
macrofauna. TABLE 2. Two-factorial Per-MANOVA for swine wastewater and soybean crop with 4999
permutations on the ecological indices of soil meso- and macrofauna obtained in the
2010–2011 and 2011–2012 crop seasons. Source of variation
DF. SS
MS
F
p-value
SW
7
0.22575
0.3225E−01
1.0869
0.3966
Crop
1
0.21398
0.21398
7.2115
0.0082
SW × Crop
7
0.36370
0.5196E−01
1.7511
0.1244
Residual
32
0.94850
0.2967E−01
Total
47
1.7529
DF – degrees of freedom; SS – sum of squares; MS – mean square; SW – swine wastewater; SW × Crop – interaction between SW
and Crop. Two-factorial Per-MANOVA for swine wastewater and soybean crop with 499
permutations on the ecological indices of soil meso- and macrofauna obtained in th
2010–2011 and 2011–2012 crop seasons. Source of variation
DF. SS
MS
F
p-value
SW
7
0.22575
0.3225E−01
1.0869
0.3966
Crop
1
0.21398
0.21398
7.2115
0.0082
SW × Crop
7
0.36370
0.5196E−01
1.7511
0.1244
Residual
32
0.94850
0.2967E−01
Total
47
1.7529
DF – degrees of freedom; SS – sum of squares; MS – mean square; SW – swine wastewater; SW × Crop – interaction between SW
and Crop. CCA between treatments and values of soil physicochemical variables is shown in Figure 3A. According to the Monte Carlo test, with only 5% significance, only the axis 1 (CC1, explanation of
43%, p-value = 0.01) can be assessed. CC1 confirms the results obtained by PCA (Figure 2) and
PerMANOVA (Table 2), demonstrating that the variability between crops is higher than the
variability between treatments. RESULTS AND DISCUSSION PC1 is composed by Simpson’s diversity, Shannon’s diversity, Buzz and Gibson’s uniformity,
Brillouin index, Margalef’s richness, equitability, Fisher’s alpha diversity, Chao1 richness, and
number of taxa in the positive quadrant, and by the variables dominance and Berger-Parker
dominance in the negative quadrant (Figure 2). The ecological indices that were grouped together in
the positive quadrant of PC1 indicate ecological stability (STENGER-KOVÁCS et al., 2016), being
assumed that these indices have a positive correlation with each other. However, the indices that
were grouped together in the negative quadrant of PC1 indicate ecological instability (STENGER-
KOVÁCS et al., 2016), showing a negative correlation with the indices located in the positive
quadrant of PC1. In addition, PC1 demonstrates that the 2011–2012 crop season presented a higher Simpson’s
diversity, Shannon’s diversity, Buzas and Gibson’s uniformity, Brillouin index, Margalef’s
richness, equitability, Fisher’s alpha diversity, and Chao1 richness. These indices show a greater
ecological balance during the sampled period. On the other hand, the 2010–2011 crop season
showed a higher dominance and Berger-Parker dominance, with an ecological instability in soil
meso- and macrofauna. Values of dominance index and Berger-Parker dominance were influenced
mainly by the order Collembola in both soybean crops. The dominance of the order Collembola was reported in studies conducted by TESSARO et
al. (2013) and CASTALDELLI et al. (2006). In these studies, the authors assessed the application
of different doses of SW (0, 100, 200, and 300 m3 ha−1) in plots cultivated with corn, oat, and Eng. Agríc., Jaboticabal, v.37, n.3, p.556-564, maio/jun. 2017 561 Soil meso- and macrofauna in two soybean crops after swine wastewater application soybean, in addition to a plot of native vegetation and another with only mineral fertilizers. The
high occurrence of the order Collembola is related to food habit because these animals feed mainly
on fungi associated with the crop straw despite being normally abundant in the litter (BLOUIN et
al., 2013; SIDDIKY et al., 2012). soybean, in addition to a plot of native vegetation and another with only mineral fertilizers. The
high occurrence of the order Collembola is related to food habit because these animals feed mainly
on fungi associated with the crop straw despite being normally abundant in the litter (BLOUIN et
al., 2013; SIDDIKY et al., 2012). PC2 was composed of the variable number of individuals in the negative quadrant and the
Menhinick’s richness in the positive quadrant. Eng. Agríc., Jaboticabal, v.37, n.3, p.556-564, maio/jun. 2017 RESULTS AND DISCUSSION However, it also confirms that the difference between crops was
caused by changes in soil physicochemical composition. In the 2010–2011 crop season, soil showed
higher values of organic matter (OM), Mg, sum of bases (SB), and cation exchange capacity (CEC)
whereas in the 2011–2012 crop season, soil presented higher values of organic N (Norg), base
saturation (BS), and NO3 + NO2. Therefore, comparing these results with PCA, the ecological
indices that represent the stability of soil meso- and macrofauna with diversity, equitability,
uniformity, among others, are influenced by the physicochemical variables Norg, BS, and NO3 +
NO2. Eng. Agríc., Jaboticabal, v.37, n.3, p.556-564, maio/jun. 2017 Ana P. C. Maciel, Silvio C. Sampaio, Marcelo B. Remor, et al. 562 FIGURE 3. Ordering of experimental units obtained in the 2010–2011 and 2011–2012 soybean
crops for the composition of soil meso- and macrofauna community as a function of
physicochemical variables using linear combinations of these variables (arrow size is
proportional to the variable effect) (Figure 3A). Composition of soil meso- and
macrofauna community according to physicochemical variables defined by the
Canonical Correlation Analysis (Figure 3B). In addition, CCA between orders of soil meso- and macrofauna and values of soil
physicochemical variables (Figure 3B) shows that the orders Orthoptera, Hymenoptera, Coleoptera,
and Collembola presented a positive correlation with OM, Mg, SB, and CEC whereas the orders
Astigmata, Diplopoda, Prostigmata, and Mesostigmata presented a positive correlation with Norg,
BS, and NO3 + NO2. The higher number of individuals from the orders Astigmata and Diplopoda in
the 2011–2012 crop season reduced the dominance of the order Collembola by increasing the values
of diversity, equitability, and uniformity indices (Figure 2). ZHU & ZHU (2015) and WANG et al. (2015) also observed the orders Collembola and Coleoptera positively correlated with OM and
Prostigmata, Diplopoda, and Mesostigmata positively correlated with total nitrogen concentration. Thysanoptera, Diptera, Oribatida, and Araneae showed no correlation with any soil
physicochemical variable, demonstrating that these orders were distributed equally between
treatments and crops. Soil meso- and macrofauna has a great mobility, which may have influenced some results
since sample units have an area of 1 m2, in addition to being close to each other. This fact may have
influenced the quantification of individuals since the migration rate from one sample unit to another
was not measured. CONCLUSIONS Doses of swine wastewater between 0 and 300 m3 ha−1 associated with mineral fertilization do
not present effects on ecological indices of soil meso- and macrofauna. Therefore, these doses do
not present perceptible ecotoxicological effects to organisms. However, periodic applications over
time change soil physicochemical variables, which influence soil community composition and may
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Digital Media-based Health Intervention on the promotion of Women’s physical activity: a quasi-experimental study
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BMC public health
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© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Peyman et al. BMC Public Health (2018) 18:134
DOI 10.1186/s12889-018-5025-5 Peyman et al. BMC Public Health (2018) 18:134
DOI 10.1186/s12889-018-5025-5 Digital Media-based Health Intervention on
the promotion of Women’s physical activity:
a quasi-experimental study Nooshin Peyman1, Majid Rezai-Rad2, Hadi Tehrani1*, Mahdi Gholian-Aval1, Mohammad Vahedian-Shahroodi1
and Hamid Heidarian Miri1 Abstract Background: Technological advances have caused poor mobility and lower physical activity among humankind. This study was conducted to assess the impact of a digital media-based (multi-media, internet, and mobile phone)
health intervention on promotion of women’s physical activity. Methods: In this quasi-experimental study, 360 women were divided into case and control groups. The digital
media-based educational intervention was conducted in two months in the case group electronically, using mail
and Internet and telephone platforms. Physical activity was measured using International Physical Activity Questionnaire
(IPAQ) that estimated women’s physical activity rate in the previous week. Data was analyzed using descriptive and
analytical statistics (ANOVA, chi-square, paired and independent t-tests) using SPSS 20. Results: The mean score of knowledge, attitude and level of physical activity in the control group were not significantly
different before and after the intervention. While in the case group, this difference before and after the intervention was
significant (p < 0.001), and mean scores of the above-mentioned factors increased after the intervention. Conclusions: Using innovative and digital media-based health education can be effective in improving health-
based behavior such as physical activity. Therefore, it seems necessary to develop user-based strategies and
strengthen the behavioral change theories and hypotheses based on digital media for effective influence on behavior. Trial registration: Iranian Registry of Clinical Trials (IRCT), IRCT20160619028529N5. Registered December 24, 2017
[retrospectively registered]. Keywords: Health behavior, Health promotion, Physical activity, Women, Health education, Fitness, International
physical activity questionnaire (IPAQ) * Correspondence: tehrani.hadi420@gmail.com
1Social Determinants of Health Research Center, Mashhad University of
Medical Sciences, Mashhad, Iran
Full list of author information is available at the end of the article Sample According to the following formula, considering type 1
error of 0.05 and power of 80%, and assuming 15% score
difference before and after the intervention, 180 women
were calculated as the sample size, and the same number
as the control group. Therefore, 360 women were
recruited. A possible way to enhance self-care behaviors and in-
crease participants’ involvement in health-related behav-
iors is the use of digital media, such as the Internet,
mobile phone technology, etc. [9]. Thus, in an attempt
to increase current rates of physical activity in women,
digital media can be used as an effective mechanism
[10]. These digital media are promising because of
potential for scalability, low cost, use in multiple settings
including
middle-
and
low-income
nations,
and
opportunities for real-time modifications and improve-
ments [9]. z 1−α
2
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2p 1−p
ð
Þ
p
þ zð1−β
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
p0 1−p0
ð
Þ þ p1 1−p1
ð
Þ
p
2
p1−p0
ð
Þ2
D The members of the intervention group were selected
from 4 health centers in Kerman. The samples were
randomly selected from a list of women visiting the
health centers and then the intervention was performed. Inclusion criteria were: being older than 18 years, having
a mobile phone and the ability to use it, access to the
internet, ability to use a computer and the internet, and
willingness to participate. The invitation to participate in
the study was sent electronically and/or by mobile to
those who had the conditions to participate in the study. Given that centers were governmental, all people partici-
pated in the study willingly (Fig. 1). According to statistics provided by the International
Telecommunication Union (ITU) in 2001, there were
more than 20,000 health-related sites, and Internet users
were estimated as 500 million people. In 2013, this
union estimated Internet users as 2.3 billion, which is
more than one-thirds of the world’s population [11]. This rate was also estimated to be 3.2 billion Internet
users in 2015 [12]. Accordingly, health education and
promotion researchers throughout the world are cur-
rently trying to explore innovative ways based on the
internet and other digital media to increase the efficacy
of their interventions [13]. The study of Vollum in 2014
[14] showed that social interaction can be increased and
enhanced by the use of social media in educational set-
tings. Design This was a quasi-experimental study. Its population was
women who referred to health centers in Kerman, Iran. At the time of the study, there were 8 active health cen-
ters in Kerman, in which 4 were randomly selected as
intervention centers and 4 as control ones. Self-care behaviors, especially during physical activity,
are recognized as important factors in maintaining an
active lifestyle. Various studies have demonstrated that
self-care behaviors are associated with promotion of
health, increased physical activity, and weight loss [8]. Sample Vollum’s study [14] also showed that social media
have already been using current programs of health/
wellness outside the K-12 system. Background physical activity (i.e., about 60–75 min per day) elimi-
nates the increased mortality risks associated with high
total sitting time [3]. For various reasons, nowadays people are having more
sedentary lifestyle. Physical activity associated with work,
home, and transportation has been decreased due to
technological advancements and social changes [1]. Increased working hours has led to having less time per-
forming physical activity Paying less attention to a more
natural life and spread of urbanization have also attrib-
uted to reduced physical activity [2]. Ekelund et al. in
2016 showed that high levels of moderate intensity Physical activity is a viable strategy that can affect non-
communicable diseases. In 2005, 35 million deaths oc-
curred
due
to
non-communicable
chronic
diseases
(NCDs) worldwide, comprising 60% of all deaths and 47%
of the diseases [4]. According to a World Health
Organization (WHO) report, these figures will reach 73%
(3/4 of all deaths) and 60% of the burden of diseases by
2020. This is even more serious in middle and low-income
countries, and has turned into an epidemic that involves
about 80% of deaths [5]. Ding et al. in 2016 showed that * Correspondence: tehrani.hadi420@gmail.com
1Social Determinants of Health Research Center, Mashhad University of
Medical Sciences, Mashhad, Iran
Full list of author information is available at the end of the article Peyman et al. BMC Public Health (2018) 18:134 Page 2 of 7 Page 2 of 7 “low-income and middle-income countries share the lar-
gest disease burden from physical inactivity” [6]. social lifestyle, the present study was conducted to assess
the impact of digital media-based intervention (multi-
media, internet, and mobile phone) to promote women’s
physical activity. The prevalence of obesity among women has been
increased. This increase was due to the changes in life-
styles, increasing mechanization of tasks and dramatic
decrease in physical activity. Benefits and impact of
regular physical activity in promoting health are well-
recognized; therefore, interventions should be designed
to encourage women to adopt, maintain and enhance
such health behaviors [7]. Measures Body mass index (BMI) was calculated using the respon-
dents’ reported height and weight. Physical activity was
measured
using
the
International
Physical
Activity
Questionnaire (IPAQ) which estimates women’s physical
activity rate in regarding MET-minutes/week [16]. A
separate questionnaire was used to investigate the know-
ledge and attitude. This questionnaire consisted of 12
questions. Six multiple choice questions were used to
measure
knowledge
and
six
five-point
Likert
style
(Completely
agree,
no
idea,
disagree,
completely
disagree) questions were used to measure attitudes. After preparing the knowledge and attitude question-
naire, content validity and face validity were performed
to test the reliability of the instrument. A literature
review was performed and experts and professors related
to the subject of the study were consulted to edit the These media have some advantages including: avail-
ability at any time or place, providing efficient content
and appropriate interaction with the user. Therefore,
these media provide a good platform for people to moni-
tor their health behaviors such as monitoring the level of
physical activity to improve health [15]. Therefore, it
seems that digital media-based intervention can increase
physics activity. Thus, given the highly important role of regular phys-
ical activity in improving women’s life quality, and
valuable women’s role in forming an active family and Peyman et al. BMC Public Health (2018) 18:134 Page 3 of 7 Fig. 1 Flow chart of the study groups comparison was performed using Independent
sample t-test. groups comparison was performed using Independent
sample t-test. questionnaire. When the face validity of the instrument
was verified, by using the content validity index (CVI)
and content validity ratio (CVR), the questionnaire was
sent to 10 experts in public health and health informa-
tion technology; their comments and view were used to
eliminate possible defects of the questionnaire. To test
the validity of the questionnaire, Cronbach’s alpha coeffi-
cient was used. The questionnaire was filled-out by 20
women referring to health centers in Kerman, Iran and
was filled-out again after 2 weeks. The Cronbach’s alpha
coefficient of internal consistency of the questionnaire
was estimated between 82% and 88% and its interclass
correlation coefficient was 71–77%. Intervention The unique characteristic of this study was its digital
media-based electronic educational intervention. In the
first step, the research team explained the importance
and the objectives of the study to attract women’s
cooperation. Moreover, women were briefed on how to
answer the questions using a completed typical ques-
tionnaire, and further explanations were also first given
if needed. Data was collected at the first stage (pre-test),
simultaneously, in all 8 centers. When the pretest was completed, media-based educa-
tional intervention program was performed for the inter-
vention group. Virtual space in this study was used in
the following way. An educational website related to
physical activity was designed with different sections. Besides different educational stuff related to physical
activity, the advantages of such activities and regular ex-
ercises and educational films were also included. In Results The demographic and socioeconomic characteristics of
the women are presented in Table 1. The two groups
were not significantly different regarding demographic
characteristics except for weight (Table 1). Before the intervention, women’s mean weight was
63.022 kg in the control group and 67.288 kg in the case
group; there was a significant difference between the two
groups (p < 0.05). However, after the intervention, mean
weight decreased to 66.388 kg in the case group, but in-
creased to 63.07 kg in the control group (p < 0.001). Before the intervention, no significant difference was
found regarding other demographic characteristics. addition to watch films on this site, women could down-
load the films on regular mobile phones and use it. The
site also had an electronic section in which women
could assess their physical activity and BMI online and
therefore, they were encouraged to self-monitor their
physical activity. Due to the Iranian culture and religious
beliefs of Muslim women and therefore restrictions of
physical activity for women in every place, a section of
the site was assigned to introduce suitable women-only
places devoted to physical activity with photos and its
characteristics, such as women-only parks, female-only
sessions, female-only gyms and sport complexes, etc. To
that end, an educational CD with several chapters was
prepared in an auto-run format according to the latest
physical activity topics by the Ministry of Health and
Medical Education of Islamic Republic of Iran. This CD
was available for women participating in the study. Furthermore,
an
educational
website
on
women’s
physical activity was designed with different sections. In
addition to various trainings about physical activity and
the benefits of regular exercise in the website, a page
was designed for educational video clips, enabling
women to watch videos, or download them on their
mobiles. In the chat room section, participants could
exchange information. Before the intervention, BMI was 24.353 kg/m2 in the
control group and 25.522 kg/ in the case group; the differ-
ence between the two groups was significant (p = 0.028). After the intervention, BMI increased to 24.404 kg/m2 in
the control group, but the increase was not significant
(p = 0.664) based on paired t-test. In the case group, mean
BMI decreased to 25.186 kg/m2, which was statistically
significant based on paired t-test (p < 0.001). Analysis Data was then analyzed using descriptive (frequency dis-
tribution, mean and standard deviation) and analytical
statistics such as ANOVA, paired and independent
t-tests by SPSS20. A paired t-test was conducted to
compare changes before and after the intervention. Two Peyman et al. BMC Public Health (2018) 18:134 Page 4 of 7 Page 4 of 7 Table 1 Frequency Distribution of Personal Details among Control
and Case Groups
Variable
Control group
Case group
p
Marital status
df = 1
p** = 0.147
Single
68(37.2%)
54(30%)
Married
113(62.8%)
126(70%)
Education level
df = 3
p** = 0.638
High school
68(37.8%)
58(32.2%)
Associate Degree
17(9.4%)
22(12.2%)
BSc. 77(42.8%)
79(43.9%)
MSc. & above
18(10%)
21(11.7%)
Age
31.933 ± 9.458
33.411 ± 9.010
p* = 0.130
Height (cm)
161.39 ± 7.488
162.38 ± 5.738
p* = 0.159
Weight (kg)
63.022 ± 12.366
67.288 ± 11.958
p* < 0.001
Body Mass Index (BMI)
24.353 ± 5.576
25.522 ± 4.370
p* < 0.05
** Pearson Chi-Square; * Independent t test Mobile phone numbers of participants and people
whose views had influence on the participants’ physical
activity (i.e. supporters) were also registered in the pretest. Then, SMS messages about the importance of physical
activity were sent daily to supporters and participants. Six
months after intervention, the impact of interventions was
assessed and data collected using the same tools. Results The results showed no significant differences between
the two groups in terms of knowledge, attitude, and phys-
ical activity before the intervention. However, according to
independent t-test, the difference between the two groups
was significant after the intervention (p < 0.05), and mean
scores of the above- mentioned scales increased among
the intervention group (Table 2). The mean score of knowledge, attitude and level of
physical activity in the control group was not signifi-
cantly
different
before
and
after
the
intervention
(Table 3). While in the case group, this difference before
and after intervention was significant (p < 0.001), and
mean scores of the above-mentioned scales increased
after the intervention (Fig. 2). Table 2 Mean and Standard Deviation of Women’s Knowledge, Attitude, and Physical Activity among Control and Case Groups before
and after Intervention
Variable
Mean and Standard Deviation Before Intervention
Mean and Standard Deviation After Intervention
Control group
Case group
p-value
Control group
Case group
p-value
Knowledge
0.8156 (±0.0600)
0.9611 (±0.641)
p* = 0.099
0.8636 (±0.0744)
6.116 (±0.0600)
p* < 0.000
Attitude
16.15
(±2.54)
16.16 (±3.412)
p* = 0.136
16.70
(±2.87)
21.10 (±2.747)
p* < 0.001
Physical activity (MET/min)
838.44 (±96.781)
992.17 (±83.302)
p* = 0.229
881.82 (±90.482)
3604.32 (±271.195)
p* < 0.001
*Independent t test Peyman et al. BMC Public Health (2018) 18:134 Page 5 of 7 Table 3 Mean and Standard Deviation of Women’s Knowledge, Attitude, and Physical Activity before and after Intervention among each of
the Control and Case Groups
Variable
Control Group
SD ± M
Case Group
SD ± M
Before Intervention
After Intervention
p*
Before Intervention
After Intervention
p*
Knowledge
0.816 (±0.0600)
0.8636
(± 0.0744)
p* = 0.586
0.9611 (±0.641)
6.116 (±0.0600)
p* < 0.001
Attitude
16.65 (± 2.54)
16.70
(± 2.87)
p* = 0.859
16.15
(± 3.41)
21.10 (± 2.74)
p* < 0.001
Physical activity
838.44 (± 96.781)
881.82
(± 90.482)
p* = 0.566
992.17
(± 83.302)
3604.32
(± 271.195)
p* < 0.001
*Paired t test Table 3 Mean and Standard Deviation of Women’s Knowledge, Attitude, and Physical Activity before and after Intervention among each of
the Control and Case Groups Table 3 Mean and Standard Deviation of Women’s Knowledge, Attitude, and Physical Activity before and after Intervention among each of
the Control and Case Groups Generally, before the intervention, control and case
groups were similar regarding their demographic charac-
teristics. Results The results showed a significant increase of
physical activity of women who used educational multi-
media and websites and received daily text messages,
compared to those in the control group. This indicates a
positive impact of media, as educational interventions,
on health promotion. Table 4 indicates that the effects of the intervention,
time and the interaction of time and intervention are
statistically significant for knowledge, attitude and phys-
ical activity. However, as it is illustrated in Table 3 the
score of knowledge, attitude and level of physical activity
are not considerably changed over two measurements
among controls group. This could be explained by the
significant
effect
of
interaction
between
time
and
intervention that emphasizes the effect of time differs
noticeably between cases and controls. Ornes and Ransdell [17] also investigated the effect
of web-based education on female university students’
physical
activity
who
received
education
through
internet for 4 weeks. By the end of the study, mean
number of steps per day had increased by 38.8%
among the group that received education through the
internet; which indicates the positive effect of using
internet and web on physical activity. The result of
this study concurs with the results of Ornes and
Ransdell’s investigation. Strengths and limitations g
Strengths of this study were using virtual space and mo-
bile phones for promotion of physical activity that
provided the space for exchanging ideas and suggestions
by participants. Limitations of the present study were
low speed and narrow internet bandwidth and, some-
times, internet disconnection. Another limitation of this
study was that the study was conducted only among
women, thus findings from this study cannot be general-
ized to the general population. In addition, given that
data collection was based on self-reporting there was the
possibility of bias in a more favorable response and
moreover, physical activity was not objectively assessed
via a physical activity assessment or activity trackers. Knowledge, attitude and behavior in our daily lives are
intertwined with each other. Changes in knowledge
cause changes in attitude. Change in attitude also causes
changes in behavior. Educational programs with using
digital media, such as internet, computer software, and
mobile phone-based ones are effective in improving
knowledge and attitudes to more physical activity [17] . Palmer et al. [20] also conducted a web-based inter-
vention among students. Palmer et al. study showed
students’ increased attitude and knowledge about phys-
ical activity, and indicated the positive effect of internet-
based intervention on their knowledge and attitude. Knowledge of the risks and benefits of lifestyle-
associated behaviors are the prerequisites of performing
a behavior. If people lack the relevant knowledge, they
will not accept reasons for enduring difficulties associ-
ated with that behavior [21]. Digital media are capable of
providing information in the shortest possible time with
maximum efficiency, and have a huge impact on know-
ledge, literacy, and attitudes of their audience [22]. Abbreviations Abbreviations
BMI: Body mass index; IPAQ: International Physical Activity Questionnaire Acknowledgements g
We express our gratitude to the management of Kerman University of Medical
Sciences (KUMS) and its deputy of health, personnel of medical centers and all
those who contributed to this study. Conclusion It seems that media-based interventions (multimedia,
internet, and mobile phone) can positively affect physical
activity of women. Therefore, it seems essential to
develop user-based strategies and strengthen behavioral
change theories and hypotheses based on digital media
by developing virtual and digital media use in the
community. Another important result of the present study was
about BMI variations before and after the intervention. Although two groups’ BMI showed a significant differ-
ence before and after the intervention, BMI had a sig-
nificant drop in the case group, after the interventions,
and a slight increase in the control group. Thus, digital
media caused increasing physical activity, which de-
creased in weight and an improvement in BMI in the
case group. Discussion This study was conducted to assess the impact of digital
media-based (multimedia, internet, and mobile phone)
intervention and education on women’s physical activity. Use of cyberspace and digital technology in a specific
group (Muslim women) was one of the most prominent
features
of
this
study
compared
to
other
studies
conducted in this area especially in Iran. With rapid growth of digital media, including internet
and mobile phones, and increasing number of people Fig. 2 Mean of Women’s Physical Activity before and after Intervention in Control and Case Groups g. 2 Mean of Women’s Physical Activity before and after Intervention in Control and Case Groups Peyman et al. BMC Public Health (2018) 18:134 Page 6 of 7 Table 4 Results from repeated measure analysis of variance for knowledge, attitude physical activity
Knowledge
Attitude
Physical Activity
Source
F
P-value
Adjusted R-squared
F
P-value
Adjusted R-squared
F
P-value
Adjusted R-squared
Model
10.44
0.0000
0.8266
2.44
0.0000
0.4222
2.91
0.0000
0.4893
intervention
864.49
0.0000
68.19
0.0000
47.91
0.0000
time
1071.5
0.0000
144.59
0.0000
94.52
0.0000
intervention × time
1034.37
0.0000
150.19
0.0000
85.13
0.0000 the case group and 66% of the participants recom-
mended the use of digital media-based programs to their
friends. Bennett et al. [24] also showed that internet-
based weight loss interventions can be useful. In their
study, they used interventions based on the internet and
mobile phone for 12 weeks, which led to weight loss in
the group that received education through the internet
and mobile phone. Furthermore, weight loss was more
prominent in participants who used more internet
interventions. with access to such media use of these media seems
necessary
to
encourage
physical
activity
[18]. The
strength of these media, compared to print media and
face-to-face intervention, is educating large numbers of
people at low costs [19]. In the present study, in addition to a significant in-
crease in physical activity in the case group, women’s
knowledge and attitude also significantly increased after
the intervention; while no increase was observed in the
control group. This shows that digital media, such as
internet, computer software, and mobile phone-based
programs, encourage women to perform physical activ-
ity, and increase their knowledge and attitude. Funding No financial support was received for this study. In a study performed by Cavallo et al. [23] to assess
the effect of social media-based intervention on physical
activity, physical activity and social support increased in Competing interests 18. Chou W-yS, Prestin A, Lyons C, Wen KY. web 2.0 for health promotion:
reviewing the current evidence. Am J Public Health. 2013;103(1):e9–e18. The authors declared no competing interests. 19. Hieftje K, Edelman E, Camenga DR, Fiellin LE. Electronic media–based health
interventions promoting behavior change in youth: a systematic review. JAMA Pediatr. 2013;167(6):574–80. Availability of data and materials Availability of data and materials
Data and materials can be requested from the corresponding author. Page 7 of 7 Peyman et al. BMC Public Health (2018) 18:134 Page 7 of 7 Peyman et al. BMC Public Health (2018) 18:134 Authors’ contributions 10. Marks JT, Campbell MK, Ward DS, Ribisl KM, Wildemuth BM, Symons MJ. A comparison of web and print media for physical activity promotion
among adolescent girls. J Adolesc Health. 2006;39(1):96–104. HT designed and implemented of the project. NP conducted all of the literature
searches. M V-h and H H-m were analyzed of data. M Gha prepared the first draft
of the paper. M R-r reviewed the full-texts of the papers for data extraction. All authors read and approved the final manuscript. 11. Bernhardt JM, Chaney JD, Chaney BH, Hall AK. New Me
Education. Health Educ Behav. 2013;40(2):129–32. 11. Bernhardt JM, Chaney JD, Chaney BH, Hall AK. New
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Health Nutr. 2006;9(06):755–62. Consent for publication
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and we will help you at every step: 7. Taggart J, Williams A, Dennis S, Newall A, Shortus T, Zwar N, Denney-Wilson
E, Harris MF. A systematic review of interventions in primary care to
improve health literacy for chronic disease behavioral risk factors. BMC Fam
Pract. 2012;13(1):49. 7. Taggart J, Williams A, Dennis S, Newall A, Shortus T, Zwar N, Denney-Wilson
E, Harris MF. A systematic review of interventions in primary care to
improve health literacy for chronic disease behavioral risk factors. BMC Fam
Pract. 2012;13(1):49. 8. O’Neill JR, Liese AD, McKeown RE, Cai B, Cuffe SP, Mayer-Davis EJ, Hamman
RF, Dabelea D. Physical activity and self-concept: the SEARCH for diabetes in
youth case control study. Pediatr Exerc Sci. 2012;24(4):577–88. 8. O’Neill JR, Liese AD, McKeown RE, Cai B, Cuffe SP, Mayer-Davis EJ, Hamman
RF, Dabelea D. Physical activity and self-concept: the SEARCH for diabetes in
youth case control study. Pediatr Exerc Sci. 2012;24(4):577–88. 9. Filion AJ, Darlington G, Chaput J-P, Ybarra M, Haines J. Examining the
influence of a text message-based sleep and physical activity
intervention among young adult smokers in the United States. BMC Public Health. 2015;15(1):671. 9. Filion AJ, Darlington G, Chaput J-P, Ybarra M, Haines J. Examining the
influence of a text message-based sleep and physical activity
intervention among young adult smokers in the United States. BMC Public Health. 2015;15(1):671.
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https://casesjournal.biomedcentral.com/counter/pdf/10.1186/1757-1626-2-150
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English
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The role of diclofenack on inducing of aplasia cutis congenita: a case report
|
Cases journal
| 2,009
|
cc-by
| 2,020
|
Open Acc
Case Report
The role of diclofenack on inducing of aplasia cutis congenita: a case
report
Laura Pajaziti*, Syzana Rexhepi, Ylfete Shatri-Muça and Mybera Ferizi Open Access * Corresponding author Published: 12 October 2009
Cases Journal 2009, 2:150
doi:10.1186/1757-1626-2-150
Received: 16 September 2009
Accepted: 12 October 2009
This article is available from: http://www.casesjournal.com/content/2/1/150
© 2009 Pajaziti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 16 September 2009
Accepted: 12 October 2009 Received: 16 September 2009
Accepted: 12 October 2009 © 2009 Pajaziti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central Abstract Background: Aplasia cutis congenita is a disorder where e newborn child is missing skin from
certain areas. It is a rare condition with no particular race or sex more at risk. May occur by itself
or be associated with other physical syndromes or disorders. A classification system exists for
aplasia cutis congenital consisting of 9 groups, based on the number and location of the skin defects
and the presence or absence of other malformations. Causes of aplasia congenital could be
heredity, teratogenic substances, placental infarcts, intrauterine infections, ectodermal dysplasias
etc. Diagnosis is made based on the clinical findings. Prognosis depends of the other organs
malfunction level and lesions size. Case report: Our case was an 22 months old Albanian girl, who was recommended to
dermatology for a consultation by a pediatric surgeon because of the changes she had on her
parietal part of the scalp with missing hair areas. The child has stenosis congenita ani and to her
was installed stoma. In order to investigate other accompanied anomalies of the disease, there are
made specific consults by neurologist, orthopedist, cardiologist, nephrologists and citogenetics. Conclusion: It was found out a minor visual discoordination, Sy Floppy, Digiti V superductus pedis
bill. Laxitas articularum generalisata. It was a great challenge for us to find out that during the first trimester of the pregnancy (unplanned
pregnancy), her mother used Diclofenac. Since there is limited information regarding to teratogenic effects of diclofenac, we considered it
interesting to present this case. Except sporadic cases, there are familiar cases, which are
explained by autosomal -dominant heredity [4]. Causes of
aplasia could be teratogenic substances, placental infarcts,
intrauterine infections, ectodermal displasias etc [5,6]. Introduction Aplasia cutis congenita is one of the disorders of the skin
embrional development. It is characterized by a limited
defect of the skin where the skin layers and its adnexes are
not created. This feature is rare, about 1 in 3000 alive born
children [1-3]. The disorders during the skin development can include
one or all skin layers. Sometimes the other deep structures Page 1 of 4
(page number not for citation purposes) Cases Journal 2009, 2:150 Cases Journal 2009, 2:150 http://www.casesjournal.com/content/2/1/150 The parietal part of the scalp appear two oval shaped areas
without hair, confluated, with light depth and shinny bases
Figure 1
The parietal part of the scalp appear two oval shaped
areas without hair, confluated, with light depth and
shinny bases. like fascia, ossa and dura are missing (which can be fol-
lowed by different cosmetic defects of the skin until the
deformities incompatible with life). Aplasia cutis congen-
ita is manifested by the defects of the skin mostly oval, 1-
3 cm in diameter, with localization on the parietal part of
scalp (60%) and rarely on the face and extremities. It
belongs to the group of congenital cicatricial alopecia. Aplasia cutis congenita can be associated by a lot of anom-
alies as: labium leporinum, polycystic kidneys, psycho-
motor retardation, developing disorders of the eyes,
spinal cord, ears, chromosomal aberationes, cutis mar-
morata, congenital organoid nevus on the scalp and face,
anomalies on extremities, intestines and genital organs. Based on the presence of these anomalies and their asso-
ciation with the other diseases as bullous epidermolysis,
growth and development stagnation, etc, congenital apla-
sias of the skin are classified in 9 groups. The parietal part of the scalp appear two oval shaped areas
without hair, confluated, with light depth and shinny bases
Figure 1
The parietal part of the scalp appear two oval shaped
areas without hair, confluated, with light depth and
shinny bases. The parietal part of the scalp appear two oval shaped areas
without hair, confluated, with light depth and shinny bases
Figure 1
The parietal part of the scalp appear two oval shaped
areas without hair, confluated, with light depth and
shinny bases. Diagnosis is made based on the clinical findings, and the
histopathology is not necessary. Prognosis depends of the other organs malfunction level
and lesions size. 46, XX with no changes on the chromosome number and
structure. Introduction The lesions can resolve spontaneously, but sometimes is
needed surgery operation (punch-graft-transplant). The routine laboratory examinations resulted with com-
mon values except the blood SE rate, which was increased
(60/1 h). After the antibiotic ordination (Cephalexin sir. A 125 mg), the same fall down to 15/1 h. The patient was
discharged home with recommendation to follow up the
pediatric surgeon visits. Case presentation Patient aged 22 months, Albanian female, hospitalized in
Clinic, by pediatric surgeon recommendation, because of
the changes on the scalp with missing hair areas. This
alteration persists from the birth without tendency for
shape changing. Authors' contributions LP, SR, YSHM and MF analyzed and interpreted the
patients' data. LP was a major contributor in writing the
manuscript. All authors read and approved the final man-
uscript. Conclusion Aplasia cutis congenita is very rare anomaly (1:3000). Causes of congenital aplasia could be teratogenic sub- On the parietal part of the scalp appear two oval shaped
areas without hair, confluated, with light depth and
shinny bases (Figure 1). Installed stoma (anus praeternaturalis)
Figure 2
Installed stoma (anus praeternaturalis). Her mother mentioned that during the first trimester of
the pregnancy (she did not know that she was pregnant)
has taken Diclofenac amp. (five days) because she had
abdominal pains. During pregnancy she did not have any
infectious disease. Family history is negative. In the surgery clinic the child was operated and treated
under the diagnoses: Stenosis congenita ani and was
installed stoma (Figure. 2). For associated defects in other
organs, there are made specific consults. Consultant orthopedist concluded the digiti V superduc-
tus pedis bilateralis, laxitas articularum generalisata and
Sy Floppy (Figure 3a and Figure 3b).Consultant neurolo-
gist indicated minor visualmotor discoordination. Cardi-
ologists and nephrolog consulting resulted on the
common stage. Citogenetic report has shown Cariotype Installed stoma (anus praeternaturalis)
Figure 2
Installed stoma (anus praeternaturalis). Page 2 of 4
(page number not for citation purposes) http://www.casesjournal.com/content/2/1/150 Cases Journal 2009, 2:150 a and b: Digiti V superductus pedis bill
Figure 3
a and b: Digiti V superductus pedis bill. Laxitas articularum generalisata Sy Floppy. A
B A A B a and b: Digiti V superductus pedis bill
Figure 3
a and b: Digiti V superductus pedis bill. Laxitas articularum generalisata Sy Floppy. g
p
p
g
a and b: Digiti V superductus pedis bill. Laxitas articularum generalisata Sy Floppy. Diclofenac used in early trimester of pregnancy might
induce those abnormalities and considered reasonable to
present this case. Diclofenac used in early trimester of pregnancy might
induce those abnormalities and considered reasonable to
present this case. stances [7]. Diclofenac is a non-steroidal anti-inflamma-
tory drug (NSAID), commonly used by reproductive age
women for the treatment of variety of conditions. NSAID
inhibit the biosynthesis of prostanoids. Women may inci-
dentally become pregnant while receiving NSAID therapy
(unplanned pregnancy) [8]. The diclofenac crosses the
placenta readily in the first trimester of human pregnancy
resulting in a fetal diclofenac concentration. which is the
same as the maternal serum concentration [9]. The drug
may also accumulate in fetal tissue with time [9]. Conclusion Usually
diclofenac is given in multiple doses, so the possibility to
reach teratogenic levels of fetal tissue concentrations is
real in patients who are taking diclofenac. In our case the
pregnant women received diclofenac 75 mg per day, five
days. Diclofenac has also been shown to inhibit implan-
tation and embryogenic development in rats when given
on gestation day 5 [10]. A positive association between
use of NSAID during pregnancy and miscarriages was
reported by a study [11]. However, information regarding
teratogenecity of NSAID during the critical period of org-
anogenesis is lacking. Because aspirin and other NSAID
share a similar mechanism of action (inhibition of pros-
taglandin synthesis) it was postulated that NSAID might
induce congenital abnormalities when given during the
critical period of organogenesis [12]. Abbreviations NSAID: non-steroidal anti-inflammatory drug; Sy: Syn-
drome. Competing interests The authors declare that they have no competing interests. Consent Written informed consent was obtained from the patient's
mother for publication of this case report and accompany-
ing images. A copy of the written consent is available for
review
by
the
Editor-in-Chief
of
this
journal. Acknowledgements The authors would like to acknowledge Consultant Orthopedist, Cardiol-
ogists and Nephrology consulting, Neurologist, and Cytogenetist who con-
tributed on provided information about the patient. We present patient with aplasia cutis congenita accompa-
nied by stenosis congenita ani, digiti V superductus pedis
bill., laxitas articularum generalisata-sy Floppy and minor
visualmotor discoordination. Based on mentioned stud-
ies and real clinical situation we concluded that References
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Braun-Falco O, Plewig G, Wolf HH, et al.: Dermatology. 2nd edi-
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2.
Karadagliæ D, et al.: Dermatologija. Beograd 2000.
3.
Mark A, Crowe MD: Aplasia cutis congenita. 2006. References References
1. Braun-Falco O, Plewig G, Wolf HH, et al.: Dermatology. 2nd edi-
tion. Springer; 1999. 2. Karadagliæ D, et al.: Dermatologija. Beograd 2000. 3. Mark A, Crowe MD: Aplasia cutis congenita. 2006. 1. Braun-Falco O, Plewig G, Wolf HH, et al.: Dermatology. 2nd edi-
tion. Springer; 1999. Page 3 of 4
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5. White G Levene's color atlas of Dermatology. 2nd edition. Mosby-Wolfe; 1997. 6. Lipozencic J, et al.: Dermatovenerologija. Medicinska naklada
Zagreb 1999. g
7. Dobriæ I, et al.: Dermatovenerologija. Zagreb 2005. 8. Chan LY, Chiu PY, Siu SSN, Lau TK: A study of diclofenac -
induced teratogenicity during organogenesis sing a whole rat
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10. Carp HJA, Fein A, Nebel L: Effect of diclofenac on implantation
and embryonic development in the rat. Eur J Obstet Gynecol
Reprod Biol 1988, 28:273-277. 11. Nielsen GL, Sorensen HT, Larsen H, et al.: Risk of adevrse birth
outcome and miscarriage in pregnant users of non-steroidal
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12. Klein KL, Scott WJ, Clark KE: A possible mechanism of aspirin
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scientist can read your work free of charge
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Evaluating the clinical effectiveness of the NHS Health Check programme: a prospective analysis in the Genetics and Vascular Health Check (GENVASC) study
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BMJ open
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cc-by
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ABSTRACT To cite: Debiec R, Lawday D,
Bountziouka V, et al. Evaluating
the clinical effectiveness
of the NHS Health Check
programme: a prospective
analysis in the Genetics
and Vascular Health Check
(GENVASC) study. BMJ Open
2023;13:e068025. doi:10.1136/
bmjopen-2022-068025
►Prepublication history and
additional supplemental material
for this paper are available
online. To view these files,
please visit the journal online
(http://dx.doi.org/10.1136/
bmjopen-2022-068025). Received 14 September 2022
Accepted 18 April 2023 To cite: Debiec R, Lawday D,
Bountziouka V, et al. Evaluating
the clinical effectiveness
of the NHS Health Check
programme: a prospective
analysis in the Genetics
and Vascular Health Check
(GENVASC) study. BMJ Open
2023;13:e068025. doi:10.1136/
bmjopen-2022-068025 Original research Open access BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 202 STRENGTHS AND LIMITATIONS OF THIS STUDY Objective The aim of the study was to assess the clinical
effectiveness of the national cardiovascular disease (CVD)
prevention programme—National Health Service Health
Check (NHSHC) in reduction of CVD risk. on October 23, 2024 by guest. Protec
http://bmjopen.bmj.com/
May 2023. Downloaded from ⇒Prospective observation of large cohort of pa-
tients from 147 primary care practices undergoing
National Health Service Health Check (NHSHC). ⇒Purposefully built, comprehensive database. Design Prospective cohort study. Setting 147 primary care practices in Leicestershire and
Northamptonshire in England, UK. ⇒Possible selection bias—individuals volunteering to
take part in the study might represent more ‘proac-
tive’ subgroup of NHSHC attendees. Participants 27 888 individuals undergoing NHSHC with
a minimum of 18 months of follow-up data. ⇒Larger than national average proportion of non-
white ethnicities representing population structure
of the region. ►Prepublication history and
additional supplemental material
for this paper are available
online. To view these files,
please visit the journal online
(http://dx.doi.org/10.1136/
bmjopen-2022-068025). Outcome measures The primary outcomes were NHSHC
attributed detection of CVD risk factors, prescription of
medications, changes in values of individual risk factors
and frequency of follow-up. ⇒The performed analysis focused on cardiovascular
disease risk factors recorded in the primary care re-
cord but has not covered influence of NHSHC on life-
style: physical exercise, diet or alcohol consumption. Results At recruitment, 18% of participants had high CVD
risk (10%–20% 10-year risk) and 4% very high CVD risk
(>20% 10-year risk). New diagnoses or hypertension (HTN)
was made in 2.3% participants, hypercholesterolaemia in
0.25% and diabetes mellitus in 0.9%. New prescription
of stains and antihypertensive medications was observed
in 5.4% and 5.4% of participants, respectively. Total
cholesterol was decreased on average by 0.38 mmol/L
(95% CI −0.34 to −0.41) and 1.71 mmol/L (−1.48 to
−1.94) in patients with initial cholesterol >5 mmol/L and
>7.5 mmol/L, respectively. Systolic blood pressure was
decreased on average by 2.9 mm Hg (−2.3 to −3.7),
15.7 mm Hg (−14.1 to −17.5) and 33.4 mm Hg (−29.4 to
−37.7), in patients with grade 1, 2 and 3 HTN, respectively. About one out of three patients with increased CVD risk
had no record of follow-up or treatment.i Received 14 September 2022
Accepted 18 April 2023 Evaluating the clinical effectiveness of
the NHS Health Check programme: a
prospective analysis in the Genetics and
Vascular Health Check
(GENVASC) study on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from Radoslaw Debiec ,1 Daniel Lawday,1 Vasiliki Bountziouka,1,2 Emma Beeston,1
Chris Greengrass,1 Richard Bramley,1 Sue Sehmi,1 Shireen Kharodia,1
Michelle Newton,1 Andrea Marshall,1 Andre Krzeminski,3 Azhar Zafar,4,5
Anuj Chahal ,6 Amardeep Heer,7 Kamlesh Khunti,4 Nitin Joshi,8 Mayur Lakhani,9
Azhar Farooqi,9 Riyaz Patel ,10 Nilesh J Samani1 Received 14 September 2022
Accepted 18 April 2023 Original research Original research GENVASC study GENVASC is a large observational study run in conjunc-
tion with Clinical Commissioning Groups and Primary
Care practices in UK and coordinated by the NIHR
Leicester Biomedical Research Centre. The GENVASC
study recruited multiethnic individuals aged 40–74 years,
attending the NHSHC at any of the 147 participating
primary care practices in Leicestershire and Northamp-
tonshire, UK (online supplemental figure 1). The main
aim of the study is to evaluate the additional clinical value
of a PRS on top of traditional risk scores in prediction of INTRODUCTION Cardiovascular disease (CVD) is a leading
cause of premature morbidity and mortality
in England.1 2 To address this, in 2009 the
UK government introduced first in the world
rolling national CVD prevention programme,
delivered by the National Health Service
(NHS). Now known as the NHS Health Check
(NHSHC), the programme aims to identify
and treat the main risk factors driving CVD,
among people aged 40–74 years, who were
otherwise well.3 It was estimated through
economic modelling that NHSHC could
prevent over 1600 myocardial infarctions
and strokes and 650 premature CVD deaths
annually.4 Cardiovascular disease (CVD) is a leading
cause of premature morbidity and mortality
in England.1 2 To address this, in 2009 the
UK government introduced first in the world
rolling national CVD prevention programme,
delivered by the National Health Service
(NHS). Now known as the NHS Health Check
(NHSHC), the programme aims to identify
and treat the main risk factors driving CVD,
among people aged 40–74 years, who were
otherwise well.3 It was estimated through
economic modelling that NHSHC could
prevent over 1600 myocardial infarctions
and strokes and 650 premature CVD deaths
annually.4 r 23, 2024 by guest. Protected by copyright. For numbered affiliations see
end of article. Correspondence to
Dr Radoslaw Debiec;
rmd24@leicester.ac.uk
© Author(s) (or their
employer(s)) 2023. Re-use
permitted under CC BY. Published by BMJ. guest. Protected by copyright. Conclusions Majority of patients identified with increased
CVD risk through the NHSHC were followed up and
received effective clinical interventions. However, one-third
of high CVD risk patients had no follow-up and therefore
did not receive any treatment. Our study highlights areas
of focus which could improve the effectiveness of the
programme. The absence of randomised clinical trials
demonstrating the effectiveness of such a
programme, has continued to raise debate
about its value.5 6 Instead, several studies Trial registration number NCT04417387. 1 1 Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 Open access on October 23, 2024 by gues
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from BMJ Open: first published as 10.1136/bmjopen-2022-0680 subsequent CVD events.15 Participants of the GENVASC
are followed up using primary care databases. The
collected information includes clinical diagnoses, labora-
tory and imaging tests, hospital admissions and referrals
to external services as well as medicinal prescriptions. INTRODUCTION sought to assess the effectiveness of the NHSHC using
various observational datasets, but often with conflicting
results, casting further doubt about the ability of the
programme to achieve the stated goals.7–11 p
g
g
The Genetics and Vascular Health Check study
(GENVASC) was instigated in 2012 to assess the value
of adding a polygenic risk score (PRS) for CVD to the
NHSHC for prediction of cardiovascular events. The
study recruited patients attending for the NHSHC in
147 primary care practices in two counties in England
(Leicestershire and Northamptonshire) and collected
comprehensive clinical data comprising the NHSHC
as well as subsequent follow-up visits. This dataset now
permits evaluation of the impact of NHSHC in more
detail than previously reported. In particular, the dataset
allows for detailed analysis of the sequelae of detecting
a risk factor, prescription of pharmacotherapy, clinical
follow-up and their correlation with observed reduction
in the risk factor. Specifically, we were able to fully charac-
terise the subpopulation of individuals, who attended the
initial NHSHC and were identified to have high and very
high cardiovascular risk as well as individuals with gross
elevations of individual risk factors. The main aim of our
study was to assess effectiveness of clinical interventions
and identify factors that may influence clinical and cost
effectiveness of the NHSHC. National Health Service Health Check The NHSHC is performed during a dedicated primary
care visit and involves structured assessment of CVD
risk.12 Adults aged 40–74 years, without pre-existing CVD,
diabetes mellitus, hypertension (HTN), hypercholes-
terolaemia and not taking statins are invited to attend
the programme. The 10-year risk of developing CVD is
calculated using QRISK2 score.13 The attendees receive
consultation regarding their CVD risk and individual
risk factors: body mass index (BMI), tobacco smoking,
blood pressure (BP), cholesterol level, alcohol intake,
physical activity and risk of diabetes. Patients found to
have increased overall CVD risk (QRISK2 score ≥10%)
or elevated individual risk factor values are given life-
style modification advice, pharmacological treatment14
and/or are referred to specialist services. Subjects with
QRISK2<10% are invited for re-assessment every 5 years. on October 23, 2024 by guest. Protected by copyright. j.com/ Baseline clinical observations were defined as measure-
ments taken during the NHSHC or the nearest measure-
ment taken around the date of NHSHC but within 30 days
from the date of the check. Any new diagnoses of CVD
risk factors made from the date of NHSHC to 12 weeks
after were attributed to the NHSHC visit. This 12-week
period, although arbitrary, was considered appropriate
for making clinical diagnosis and starting any treatment
and supported by the bar plot of distribution of weekly
diagnoses post-NHSHC (figure 1). 2024 by guest. Protected by copyright. Study participants For the purpose of current analysis we included 27 888
participants of the GENVASC study recruited between 17
June 2010 and 04 September 2018 who had 18 months of
follow-up data.15 Based on an estimate from Patel et al16
about the annual attendance to NHSHC in Leicestershire
and Northamptonshire, the population used for current
analysis constituted around 10% of all NHSHC attendees
in these two counties. All participants gave their consent
to access their medical records during the duration of the
study. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from Clinical variables and definitions i
For details regarding methods of assessment and
extraction of clinical and laboratory variables (see
online supplemental material). Overall, 10-year CVD
risk was defined from QRISK2 score and categorised as
low (<10%), high (10–20%) and very high (>20%).13 BP
was defined as normal if systolic blood pressure (SBP)
<140 mm Hg and/or diastolic blood pressure (DBP) <90
mm Hg; grade 1 HTN if SBP was 140–159 mm Hg and/
or DBP 90–99 mm Hg, grade 2 HTN if SBP was 160–179
mm Hg and/or DBP 100–109 mm Hg, grade 3 HTN if
SBP was ≥180 mm Hg and/or DBP≥110 mm Hg.17 Total
cholesterol (TCh) was defined as normal for values <5.0
mmol/L, high for TCh values 5.0–7.49 mmol/L and very
high for TCh≥7.5 mmol/L.18 Non-high density lipopro-
tein (non-HDL) was defined as normal if values were
<3.8 mmol/L and high for values ≥3.8 mmol/L.18 Kidney
function was considered normal if estimated glomerular
filtration rate (eGFR) ≥60 mL/min/1.73m²; chronic
kidney disease (CKD) was diagnosed if eGFR<60 mL/
min/1.73m².19 Body weight was assessed using BMI and
considered as healthy body weight if BMI<25 kg/m2 (<23
kg/m2 for South Asians), overweight for BMI from 25
to 29.9 kg/m2 (23 to 27.4 kg/m2 for South Asians) and
obese if BMI≥30 kg/m2 (>27.5 for South Asians). Outcomes of interest The assessment of the impact of the NHSHC was
performed using the following measures: 2 Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 Open access
on October 23, 2024 b
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from Open access Key characteristics of the study cohort Overall 27 888 GENVASC participants were included in
the current analysis. The median age of included partici-
pants was 51 years, 44% (n=12 322) were men, 81% (n=22
677) were of white ethnicity and 13% (n=3686) were South
Asians (online supplemental table 1). The GENVASC
population was, in general younger, had a higher propor-
tion of Asians among the ethnic minorities, and had
more participants from the extremes of the Townsend
deprivation index (quintiles 1 and 5), compared with the
NHSHC attendees across England or the general adult
population aged between 40 and 74 years, although the
differences were small (table 1). Patient and public involvement p
p
j
g
py
►Frequency of follow-up—number of clinical visits/
reviews within the specified periods. GENVASC was developed in collaboration with the NIHR
Leicester Biomedical Research Centre Cardiovascular
Public Involvement Group. The research plan and all
public facing materials were reviewed and approved prior
to ethics submission. The group was updated biannually
on the study. This was later extended to our tissue public
involvement group (The Exceed Group). ►Change in values of individual risk factors—differ-
ence between the value of a given clinical character-
istic recorded during the NHSHC and first value of
this characteristic recorded after 12 months from the
NHSHC visit. Cardiovascular risk groups At recruitment, 18% (n=5090) of participants were classi-
fied to have high CVD risk and 4% (n=1162) to have very
high CVD risk (online supplemental table 1). Patients in
the high and very high CVD risk groups were on average
older than patients in the low CVD risk groups (median
age 65, 69 and 48 years, respectively), were more like
to be male, of white ethnic origin and current smokers
(online supplemental table 1). p
Group comparisons were performed using a χ2 test for
categorical variables and the one-way analysis of variance
for the normally distributed continuous variables or the
Kruskall-Wallis for the non-normally distributed ones. The change in the levels of individual risk factors was
calculated as the difference between the initial NHSHC
measurement and the measurement during follow-up,
after a period of 12 months. To assess the statistical signif-
icance of that comparison, the paired t-test for the contin-
uous and the McNemar test for the nominal data were
used. Open access on October 23, 2024 by guest. Protecte
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from were two-sided and Stata V.16.0 was used for all the
calculations.20 were two-sided and Stata V.16.0 was used for all the
calculations.20 ►Medication prescription and non-medical interven-
tions (eg, smoking cessation)—absolute number and
proportion of subjects started on a given therapy. Statistical analysis Categorical variables (sex, ethnicity, deprivation based
on quintiles of Townsend 2011 index (for further infor-
mation on Townsend index see Supplementary Data—
Townsend Deprivation Score), smoking status, BP
category, TCh category, renal function category, over-
weight/obesity, QRISK2 category) are shown as frequen-
cies (relative frequency) while continuous data are shown
as means and SD if normally distributed (SBP, DBP, TCh),
or median (IQR) otherwise (age, eGFR). Normality was
assessed using graphical methods (ie, histograms, P–P
and Q–Q plots). 2 en: first published as 10.1136/bmjope http://bmjopen.bmj.c
published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from 3
Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025
►Risk
factor
detection—absolute
number
and
proportion of participants with abnormal values
of BP, TCh, eGFR, BMI, current tobacco smoking
or having a clinical diagnosis of HTN, diabetes,
hypercholesterolaemia, CKD or atrial fibrillation. Both the rates of clinically coded diagnoses and the
rates of observed abnormal values were presented to
accurately assess prevalence of risk factors. Figure 1 Weekly incidence of cardiovascular disease risk factors in relation to National Health Service Health Check (NHSHC). BMI, body mass index; eGFR, estimated glomerular filtration rate. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
blished as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from en-2022-068025 on 30 May 2023. D http://bmjopen.bmj
Downloaded from Figure 1 Weekly incidence of cardiovascular disease risk factors in relation to National Health Service Health Check (NHSHC). BMI, body mass index; eGFR, estimated glomerular filtration rate. on October 23, 2024 by guest. Prote
.com/ hypercholesterolaemia, CKD or atrial fibrillation. Both the rates of clinically coded diagnoses and the
rates of observed abnormal values were presented to
accurately assess prevalence of risk factors. hypercholesterolaemia, CKD or atrial fibrillation. Both the rates of clinically coded diagnoses and the
rates of observed abnormal values were presented to
accurately assess prevalence of risk factors. ►Risk
factor
detection—absolute
number
and
proportion of participants with abnormal values
of BP, TCh, eGFR, BMI, current tobacco smoking
or having a clinical diagnosis of HTN, diabetes, Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 3 BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. D Data extraction on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
136/bmjopen-2022-068025 on 30 May 2023. Downloaded from Data about participants were extracted from the primary
care information technology systems. The primary care
databases (EMIS and SystmOne) were used to obtain
participants’ sociodemographic information (age, sex
and ethnicity), anthropometric measurements (height,
weight), information about health-related behaviour
(tobacco smoking), clinical diagnoses, blood results and
prescribed medicines. Relevant data were extracted using
NHS Numbers, SNOMED CT/DM&D and Clinical Terms
Version 3 (formerly known as the Read codes). Diagnoses and risk factor detection The majority of new clinical diagnoses of HTN, hypercho-
lesterolaemia, diabetes mellitus and CKD occurred within
the first 12 weeks following attendance to the NHSHC
(figure 1). There was also a slight increase in the rate
of new diagnoses of HTN, hypercholesterolaemia and
diabetes between 12 weeks and 6 months post-NHSHC
(figure 1). After that period the rate of new diagnoses
plateaued and remained stable through the study
(figure 1). In contrast to HTN, hypercholesterolaemia
and diabetes, the rate of new diagnoses of atrial fibrilla-
tion was similar through the study duration. 23, 2024 by guest. Protected by copyright. Subjects with no repeated values recorded for the
measured variables from after the initial NHSHC visit
to the total time of follow-up observation of 18 months
were considered as not followed up. The comparisons
between individuals without follow-up and those that
were followed up were performed with the indepen-
dent t-test and Pearson’s χ2 test for the continuous and
nominal data, respectively. A logistic regression model
was used to assess whether sociodemographic (age, sex,
deprivation, ethnicity) and clinical factors (SBP, DBP,
TCh, BMI, smoking) were associated with the absence
of follow-up. Significance level was set to 0.05, all tests guest. Protected by copyright. There was a discrepancy between the proportion of
diagnoses coded in medical records and observation of
abnormal clinical and laboratory measurements (table 2). Within 12 weeks from recruitment to the NHSHC,
abnormal BP measurements (SBP≥140 mm Hg and/or
DBP≥90 mm Hg) were recorded for 27% (n=7516) indi-
viduals while coded diagnosis of HTN was recorded for
only 2.3% (n=628). Similar pattern was observed for the Debiec R, et al. BMJ Open 2023;13:e068025. Diagnoses and risk factor detection All presented QRISK2 scores incorporate Townsend scores (available to GPs at the time of consultation). GENVASC, The Genetics and Vascular Health Check; NHSHC, National Health Service Health Check; ONS, Office for National Statistics. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/ clinical diagnosis of hypercholesterolaemia, obesity and
CKD. Particularly, 58.7% (n=16 379) and 2% (n=547) indi-
viduals were found to have TCh≥5 mmol/L and TCh≥7.5
mmol/L, respectively, while only 70 (0.25%) individuals
received a coded diagnosis of hypercholesterolaemia. Coded diagnoses of obesity and CKD were recorded for
0.5% (n=144) and 0.07% (n=19) patients, respectively. However, within the same period values of BMI meeting
criteria for obesity (≥30 kg/m2 and ≥27.5 kg/m2 for Asian
participants) and eGFR (<60 mL/min/1.73m2) were
recorded for 26.2% (n=7315) and 0.9% (n=174) patients,
respectively (table 2). Table 2 Comparison of coded diagnoses to rates of
abnormal results with 12 weeks of the NHS Health Check
Diagnoses made within 12 weeks from
NHSHC
Counts (%)
Clinical diagnosis of HTN
628 (2.3)
Abnormal BP reading (SBP≥140 mm Hg and/
or DBP≥90 mm Hg)
7516 (27)
Clinical diagnosis of hypercholesterolaemia
70 (0.25)
High total cholesterol (TCh≥5 mmol/L)
16 379 (58.7)
Very high total cholesterol (TCh≥7.5 mmol/L)
547 (2)
Clinical diagnosis of diabetes mellitus
248 (0.9)
Clinical diagnosis of obesity
144 (0.5)
BMI meeting criteria for obesity (≥30 kg/m2
and ≥27.5 kg/m2 for Asian participants)
7315 (26.2)
Clinical diagnosis of CKD
19 (0.07)
Abnormal eGFR (<60 mL/min/1.73m2)
174 (0.9)
BMI, body mass index; BP, blood pressure; CKD, chronic
kidney disease; DBP, diastolic blood pressure; eGFR, estimated
glomerular filtration rate; HTN, hypertension; NHSHC, National
Health Service Health Check; SBP, systolic blood pressure; TCh,
total cholesterol. Table 2 Comparison of coded diagnoses to rates of
abnormal results with 12 weeks of the NHS Health Check on October 23, 2024 by guest. Protected by copyright. .com/ 3, 2024 by guest. Protected by copyright. Diagnoses and risk factor detection doi:10.1136/bmjopen-2022-068025 4 Open access Table 1 Comparison of GENVASC study participants included in the current analysis to the general population of England
and large population of NHSHC attendees Table 1 Comparison of GENVASC study participants included in the current analysis to the general population of England
and large population of NHSHC attendees
ONS data* n=22 805 612
NHSHC data* n=5
102 758
GENVASC study
n=27 888
Missingness (%)
Demographic
Males, n (%)
11 200 690 (49.1)
2 311 604 (45.3)
12 327 (44.3)
–
Age bands (years)
–
40–49
7 525 814 (33)
1 951 264 (38.2)
12 256 (43.9)
50–59
7 089 322 (31.1)
1 742 003 (34.1)
8495 (30.5)
60–74
8 190 476 (35.9)
1 409 491 (27.7)
7137 (25.6)
Ethnicity, n (%)
–
White
20 383 677 (89.3)
4 067 864 (79.7)
22 677 (81.3)
Asian
1 341 580 (5.9)
368 145 (7.2)
3686 (13.2)
Black
585 756 (2.6)
148 160 (2.9)
535 (1.9)
Other ethnicity
494 599 (2.2)
142 621 (2.8)
990 (3.6)
Missing data
375 968 (7.4)
Quintiles of deprivation, n (%)
2.4
First quintile (least deprived)
4 996 212 (21.9)
1 129 670 (22.1)
8332 (29.9)
Second quintile
4 901 834 (21.5)
1 094 925 (21.5)
5867 (21.0)
Third quintile
4 707 382 (20.6)
1 027 096 (20.1)
4429 (15.9)
Fourth quintile
4 286 645 (18.8)
954 656 (18.7)
3415 (12.3)
Fifth quintile (most deprived)
3 913 539 (17.2)
893 194 (17.5)
5167 (18.5)
Missing data
–
3217 (0.1)
678 (2.4)
*Patel et al.16
†The presented Townsend scores were obtained using post codes provided by patients on consent forms (available for only a proportion of
patients). All presented QRISK2 scores incorporate Townsend scores (available to GPs at the time of consultation). GENVASC, The Genetics and Vascular Health Check; NHSHC, National Health Service Health Check; ONS, Office for National Statistics. d
-
5
s
. r
. g
n
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,
%
-
8
t
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y
%
,
,
%
t
on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
shed as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from Patel et al. †The presented Townsend scores were obtained using post codes provided by patients on consent forms (available for only a proportion of
patients). Frequency of follow-up The frequency of follow-up visits was mainly associated
with the severity of the abnormality detected during the
NHSHC. p
g
g
p
Participants in the very high CVD risk group, evidenced
reduction, on average, in the SBP and DBP levels by 7.5
(−9.4 to −5.7) and 5 (−6.0 to −3.9) mm Hg, respectively
(average time to repeated measurement 71±8 weeks) and
in median eGFR by 3 (−5.0 to −0.8) mL/min/1.73m2
(average time to repeated measurement 74±15 weeks). There were also clinically significant reductions in
TCh and non-HDL cholesterol by 0.79 (−0.91 to −0.69)
mmol/L, −0.68 (−0.83 to −0.53) mmol/L, respectively
(average time to repeated measurement 74±15 weeks). The BMI reduced on average by −0.3 (−0.52 to −0.11)
kg/m2, respectively (average time to a repeated measure-
ment 73±15 weeks) (table 4, very high CVD group). Over 18 months, patients, with grade 1, 2 and 3 HTN,
as assessed during NHSHC visit, had a median of 4 (95%
CI 2 to 9), 4 (95% CI 2 to 11) and 6 (95% CI 2 to 12)
measurements of BP, respectively. Over 18 months of
follow-up, at least a single repeated measurement of BP
was performed in 41% of patients with normal, 58% with
grade 1, 81% with grade 2 and 88.5% of patients with
grade 3 HTN, respectively. g
p
y
Over 18 months of follow-up repeated measurements
of TCh were performed in 20%, 28% and 57% of patients
with normal, high and very high TCh, respectively. 23, 2024 by guest. Protected by copyright. g
y
g
p
y
In contrast to the repeated measurements of BP and
TCh, repeated measurements of body weight were done
less frequently, with around one in four participants in
total having a record of repeated weight measurements
over the 18 months. At least a single repeated weight
measurement was recorded for 17% of overweight and
25% of obese participants over 18 months of follow-up. There was a strong association between the overall cardio-
vascular risk and repeated measurements of BMI. At least
on repeated measurement of BMI was performed for
24%, 30% and 37% of participants with low, high and very
high CVD risk, respectively. y
g
g
p
Analogous to the gradient of CVD risk, the magnitude
of the changes in the individual risk factors levels varied
according the severity of the abnormality observed for
that risk factor, during the NHSHC. Open access on October 23, 2024 by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from 18 months 4.5%, 13.5% and 28.8% of GENVASC partic-
ipants, meeting criteria for grade 1, grade 2 and grade
3 HTN, respectively were prescribed more than one
group of antihypertensive medications. The time from
the NHSHC to onset of antihypertensive medication
showed big discrepancies, however, there was a strong
trend for shorter time to pharmacological treatment with
higher category of HTN. In particular, median time from
NHSHC to first prescription was reduced from 23 weeks
for patients with grade 1 HTN, to 12 weeks for those with
grade 2 HTN and 6 weeks for those with grade 3 HTN. In addition to association with CVD risk there was an
association between prescription of statins and antihyper-
tensive medications and follow-up. Among patients with
evidence of follow-up antihypertensive medications and
statins were prescribed to 24% and 29.8% in compar-
ison to 2.2% and 5.7% of patients with no evidence of
follow-up (table 3). on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
hed as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from Change in the levels of CVD risk factors (first repeated
measurement after 12 months from the NHSHC) The magnitude of changes in the CVD risk factors during
the follow-up period were relevant to the attendant’s
initial CVD risk severity group, allocated during the
NHSHC visit, and were more evident in participants in
the very high-risk group. g
g
Similar trends were observed for the prescription of
lipid lowering medications. Statins were prescribed to
5.4% (1514) of all attendees and the proportion gradu-
ally increased to 7.1% (1984) at 18 months. The propor-
tion of patients prescribed statins increased with the
severity of CVD risk, while the median time to statin
prescription was decreased as the severity of CVD risk was
increased. Specifically, within the first 12 weeks following
the NHSHC, statins were prescribed to 1.8%, 14.2%
and 34.2% of individuals in the low, high and very high
CVD risk groups, respectively, reaching 2.8%, 18% and
39.2% at 18 months. The median time from the NHSHC
to statin prescription was 29 weeks for the low CVD risk
group, reduced to seven and 4 weeks for the high and
very high CVD risk groups, respectively. Within the first
12 weeks following the NHSHC, 5.3% of participants with
high TCh and 24.5% of participants with very high TCh
were started on statins, reaching 7.4% and 33%, at 18
months, respectively. At follow-up participants in the low CVD risk category,
had on average higher SBP and DBP by 3.6 (3.1 to 3.9)
and 1.2 (1.0 to 1.6) mm Hg, respectively (average time to
observation 74±15 weeks), and lower median eGFR by 5
(−5.6 to −4.3) mL/min/1.73m² (average time to observa-
tion 75±15 weeks). There was a minor reduction in the
TCh levels by 0.14 (−0.16 to −0.10) mmol/L, (average
time to repeated measurement 76±15 weeks) (table 4, low
CVD group). For participants in the high CVD risk group, there was
no significant change in the SBP. DBP decreased by on
average 0.9 (−1.4 to −0.40) mm Hg (average time to the
follow-up measurement 73±15 weeks), while total and
non-HDL cholesterol were decreased by 0.45 (−0.51 to
−0.39) and 0.47 (−0.55 to −0.39) mmol/L, respectively
(average time to repeated measurement 75±15 weeks). The median eGFR was lower by 3 (−4.4 to −1.6) mL/
min/1.73m² (average time to repeated measurement
74±15 weeks). There was no significant change in the
BMI at follow-up (table 4, high CVD group). Medical prescriptions and clinical interventions Medical prescriptions and clinical interventions Within the first 12 weeks following the NHSHC visit, 5.4%
(1,510) of all attendees were started on antihyperten-
sive medications, which increased to 8.4% (2337) at 18
months. Within the first 12 weeks from the NHSHC visit
antihypertensive medications were prescribed for 3.6%,
10.0% and 21.2% of participants with low, high and very
high CVD risk, respectively and 9.7%, 26.7% and 61.2%
for grade 1, 2 and 3 HTN, respectively. At 18 months,
these proportions increased to 6.4%, 14.8% and 26.2%,
for the three CVD risk groups, and to 15.4%, 36.9%
and 63.1% for the three HTN groups, respectively. At 5 Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 Frequency of follow-up Overall, individuals
with BP above 140/90 mm Hg, as measured during the
NHSHC, achieved an average reduction of 7 (−7.7 to
−6.3) mm Hg of SBP and 4.6 (−5.1 to −4.2) mm Hg of
DBP (average time to repeated observation 73±15 weeks). For grade 1, grade 2 and grade 3 HTN patients, SBP
decreased on average by 2.9 (−3.7 to −2.3), 15.7 (−17.5 guest. Protected by copyright. 6 Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 Open access Table 3 Demographic and clinical characteristics of participants with high and very high CVD risk who did not have record of
follow-up
Delta (N-Y)
P value
Follow-up
No follow-up
(95% CI)
High and very high CVD risk, n (%)
(QRISK2≥10)
4217 (67.5)
2035 (32.5)
Demographic
Male sex, n (%)
2738 (64.9)
1378 (67.7)
2.8 (0.3 to 5.2)
0.0290
White ethnic background, n (%)
3615 (85.7)
1850 (90.9)
5.2 (3.5 to 6.8)
<0.0001
Age, years (median, CI)
64.2 (63.9 to 64.4)
64.6 (64.4 to 64.9)
0.47 (0.11 to 0.83)
0.0104
Quintiles of deprivation, n (%)
First quintile
1240 (29.9)
656 (32.8)
−2.9 (−5.4 to −0.4)
0.0229
Second quintile
875 (21.1)
483 (24.1)
−3.0 (−5.3 to 0.8)
0.0077
Third quintile
707 (17.0)
305 (15.2)
1.8 (−0.1 to 3.7)
0.0809
Fourth quintile
490 (11.8)
236 (11.8)
–
1
Fifth quintile (most deprived)
841 (20.2)
323 (16.1)
4.1 (2.0 to 6.2)
0.0001
Clinical
Current smokers, n (%)
781 (18.5)
359 (17.6)
−0.9 (−0.03 to 0.11)
0.3876
Ex-smokers, n (%)
1176 (27.9)
501 (24.6)
−3.3 (−5.6 to −0.9)
0.0058
BMI, kg/m2
27.9 (5.2)
26.9 (4.7)
−1.0 (−1.0 to −0.66)
<0.0001
Systolic blood pressure, mm Hg
139.3 (17.8)
132.6 (13.5)
−6.7 (−7.0 to −5.9)
<0.0001
Diastolic blood pressure, mm Hg
82.3 (11.2)
78.7 (8.9)
−3.6 (−4.0 to 3.1)
<0.0001
Antihypertensive medications, n (%)
1012 (24.0)
44 (2.2)
−21.8 (−23.2 to −20)
<0.0001
Total cholesterol, mmol/L
5.6 (1.1)
5.5 (1.0)
−0.1 (−0.1 to −0.02)
0.0079
Non-HDL cholesterol, mmol/L
4.0 (1.0)
3.9 (0.9)
−0.1 (−0.1 to 0.01)
0.0622
Statins, n (%)
1257 (29.8)
116 (5.7)
−24.1 (−25.8 to −22.4)
<0.0001
Results are mean (SD) unless otherwise stated. BP was defined as normal if SBP<140 mm Hg and/or DBP<90 mm Hg; TCh
was defined as normal for values <5.0 mmol/L; non-HDL was defined as normal if values were <3.8 mmol/L; body weight was
considered as healthy body weight if BMI<25 kg/m2 (<23 kg/m2 for South Asians). Individuals with no record of follow-up The demographic and clinical characteristics of high and
very high CVD risk patients with unrecorded follow-up
are presented in table 3. Approximately one in three
patients with high or very high CVD risk had no recorded
evidence of a clinical follow-up in the 18-month period
following the NHSHC. Lack of clinical follow-up was asso-
ciated with male sex and white ethnic background and
lower index of social deprivation (table 3). Participants
without evidence of follow-up were more likely to be
ex-smokers, had, at the time of the NHSHC, on average
lower values of BMI, SBP and DBP than individuals who
had record of follow-up, although the differences were
small (table 3). Results from the logistic regression anal-
ysis showed that other than white ethnic background 23, 2024 by guest. Protected by copyright. There was a small reduction in the BMI with overweight
patients losing approximately 0.08 (−0.15 to −0.007) kg/
m2 and obese patients losing 0.27 (−0.37 to −0.12) kg/
m2 (average time to repeated observation 75±15 weeks)
(figure 2, bottom panel). guest. Protected by copyright. Frequency of follow-up BMI, body mass index; CVD, cardiovascular disease; HDL, high density lipoprotein; NHSHC, National Health Service Health
Check. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from Results are mean (SD) unless otherwise stated. BP was defined as normal if SBP<140 mm Hg and/or DBP<90 mm Hg; TCh
was defined as normal for values <5.0 mmol/L; non-HDL was defined as normal if values were <3.8 mmol/L; body weight was
considered as healthy body weight if BMI<25 kg/m2 (<23 kg/m2 for South Asians). BMI, body mass index; CVD, cardiovascular disease; HDL, high density lipoprotein; NHSHC, National Health Service Health
Check. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/ to −14.1) and 33.4 (−37.7 to −29.4) mm Hg, respectively
(figure 2, top panel). 60% (2275) at 18 months, while 14% of participants had
multiple records of advice for smoking cessation. At 18
months 451 (12%) participants quit smoking, according
to the GP records. A similar pattern was observed for the TCh levels, with
an average reduction of 0.38 (−0.41 to −0.34) mmol/L
and 1.71 (−1.94 to −1.48) mmol/L, respectively, for
patients with high and very high TCh (average time to
repeated measurement 75±15 weeks) (figure 2, middle
panel). Results are mean (SD) unless otherwise stated. BP was defined as normal if SBP<140 mm Hg and/or DBP<90 mm Hg; TCh
was defined as normal for values <5.0 mmol/L; non-HDL was defined as normal if values were <3.8 mmol/L; body weight was
considered as healthy body weight if BMI<25 kg/m2 (<23 kg/m2 for South Asians).
BMI, body mass index; CVD, cardiovascular disease; HDL, high density lipoprotein; NHSHC, National Health Service Health
Check. Smoking cessation During the NHSHC, 16% (3813; 19% men, 14% women)
were recorded as current smokers. Of the 3813 current
smokers, 56% (2133; 53% men, 47% women) had a record
of smoking cessation advice documented during or within
30 days of the NHSHC. This proportion increased to Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 7 on October 23, 2024 by guest. Protected by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from Open access
Table 4 Comparison of clinical characteristic of participants between NHSHC and follow-up
Parameters
NHSHC
Follow-up
Delta (FU-HC)
P value
(12–18 months)
(95% CI)
Low CVD risk (QRISK2<10)
Obesity, n=3747
Body mass index, kg/m2
28.0 (5.9)
28.0 (5.9)
0.006 (−0.07 to 0.05)
0.83
Blood pressure, n=6785
Systolic, mm Hg
125.5 (15.6)
129.1 (16.3)
3.6 (3.1 to 3.9)
<0.001
Diastolic, mm Hg
78.7 (10.2)
79.9 (10.4)
1.2 (1.0 to 1.6)
<0.001
Cholesterol, n=3233
Total cholesterol, mmol/L
5.4 (1.0)
5.3 (0.98)
−0.14 (−0.16 to −0.10)
<0.001
Non-HDL-cholesterol, n=1339
Non-HDL-cholesterol, mmol/L
3.9 (1.0)
3.7 (0.98)
−0.12 (−0.16 to −0.08)
<0.001
Median eGFR, n=2551
eGFR, mL/min/1.73m²
86 (85; 87)
81 (75; 86)
−5 (−5.6 to −4.3)
<0.0001
High CVD risk (QRISK2 10–20)
Obesity, n=1214
Body mass index, kg/m2
27.6 (5.4)
27.6 (5.7)
−0.08 (−0.20 to 0.06)
0.27
Blood pressure, n=2046
Systolic blood pressure, mm Hg
136.7 (16.5)
136.4 (16.6)
−0.3 (−1.1 to 0.53)
0.5
Diastolic blood pressure, mm Hg
81.1 (10.8)
80.2 (10.2)
−0.9 (−1.4 to −0.40)
<0.001
Cholesterol, n=1339
Total cholesterol, mmol/L
5.6 (1.1)
5.1 (1.1)
−0.45 (−0.51 to −0.39)
<0.001
Non-HDL-cholesterol, n=583
Non-HDL-cholesterol, mmol/L
4.0 (1.0)
3.6 (1.1)
−0.47 (−0.55 to −0.39)
<0.001
Median eGFR, n=1129
eGFR, mL/min/1.73 m²
82 (78; 81)
79 (78; 80)
−3 (−4.4 to −1.6)
<0.001
Very high CVD risk (QRISK2>20)
Obesity, n=354
Body mass index, kg/m2
28.7 (6.1)
28.4 (6.0)
−0.3 (−0.52 to −0.11)
0.002
Blood pressure, n=571
Systolic blood pressure, mm Hg
146.2 (20.8)
138.7 (16.3)
−7.5 (−9.4 to −5.7)
<0.001
Diastolic blood pressure, mm Hg
84.8 (12.5)
79.8 (10.4)
−5 (−6.0 to −3.9)
<0.001
Cholesterol, n=419
Total cholesterol, mmol/L
5.6 (1.1)
4.8 (1.2)
−0.79 (−0.91 to −0.69)
<0.001
Non-HDL-cholesterol, n=174
Non HDL-cholesterol, mmol/L
4.1 (1.0)
3.4 (1.1)
−0.68 (−0.83 to −0.53)
<0.001
Median eGFR, n=303
eGFR, mL/min/1.73m²
81 (79; 83)
78 (76; 80)
−3 (−5.0 to −0.8)
0.006
Results are shown as mean (SD) unless otherwise indicated. DISCUSSION We present results evaluating the clinical effectiveness of
the NHSHC in identifying, treating and monitoring indi-
viduals at high CVD risk. Our study highlights novel find-
ings which may influence implementation of strategies
for improving effectiveness of the programme. g
py
Our data indicate that the problem of patients with
increased CVD risk (QRISK2≥10) not subjected to
follow-up has not been adequately explored. A previous
survey performed in general practices in London in the
first year after implementation of the NHSHC identified
problems with implementation.24 The identified prob-
lems included non-prescribing of statins to high-risk
individuals, reluctance to refer to external services and
variable patterns of organising clinical follow-up with only
around 50% of practices organising the recommended
annual recall.24 Forster et al also reported that fewer than
52% of patients had repeated monitoring of CVD risk
factors over the 15-month period of observation.8 Several
studies reported lower than expected prescriptions of
statins to patients diagnosed with elevated TCh and/or
high CVD risk during NHSHC.8–10 A key finding from our study is the characterisation of
high and very high CVD risk patients (QRISK2≥10), who
had attended the NHSHC, but have not had any record of
clinical follow-up over 18 months and have not received
any treatment. This is a considerably large subpopula-
tion of patients and our study showed that this group has
specific social, demographic and clinical characteristics. It is also the group of patients, where preventive interven-
tions are likely to bring the largest clinical benefits and
be most cost effective. Similar observations were made
for patients with very abnormal values of isolated risk
factors—such as BP and cholesterol. These subgroups of
participants have a very high CVD risk, although it may
not be accurately estimated by QRISK2.13 This group
of patients may need multidisciplinary approach using
specialist services including genetic testing and cascade
screening, which cannot be provided in primary care
practices. on October 23, 2024 by guest. Protec
http://bmjopen.bmj.com/
0 May 2023. Downloaded from g
g
Lower than projected uptake of the NHSHC has been a
concern as the major factor influencing cost-effectiveness
of the programme.25 Recent data indicate a steady
increase in the uptake now reaching a satisfactory level
of 52%.16 26 Our study indicates that overall effectiveness
of the NHSHC can be improved by optimising delivery of
interventions reducing the CVD risk. DISCUSSION p
The second important finding relates to the level of
control of risk factors. Despite the substantial improve-
ments in both BP and TCh, their values at 18 months
remained high (figure 2). This indicates that even when
patients were started on treatment, there does not seem
to be an imperative to ensure that the risk factors are well
controlled. This may be due to several factors including
high workload of primary care practices and lack of clear
definition of primary prevention treatment targets. It is
also likely that it reflects the way primary care is incen-
tivised. Primary care practices receive a payment from
NHS once a new case is identified or a new treatment
is being initiated, rather than receiving a payment once
treatment goals have been achieved.21 Our study suggests
that this mechanism may be insufficient. Our data also
showed lack of adherence to the National Institute for
Health and Care Excellence (NICE) recommendation,
which advocates re-examination of lipid profile 3 months
after prescribing statins.14 Lack of repeated cholesterol
measurements may be one of the factors significantly
reducing uptake and continuation of statin treatment.22 on October 23, 2024 by guest. Protected by copyright. bmjopen.bmj.com/ Smoking cessation Differences in mean values or proportions between the follow-up and the
NHSHC, along with the relevant 95% CIs, are also shown. P values derived from the paired t-test for the continuous variables and the
McNemar test for the nominal paired variables. BP was defined as normal if SBP<140 mm Hg and/or DBP<90 mm Hg; TCh was defined Results are shown as mean (SD) unless otherwise indicated. Differences in mean values or proportions between the follow-up and the
NHSHC, along with the relevant 95% CIs, are also shown. P values derived from the paired t-test for the continuous variables and the
McNemar test for the nominal paired variables. BP was defined as normal if SBP<140 mm Hg and/or DBP<90 mm Hg; TCh was defined
as normal for values <5.0 mmol/L; non-HDL was defined as normal if values were <3.8 mmol/L; kidney function was considered normal if
eGFR≥60 mL/min/1.73m²; body weight was considered as healthy body weight if BMI<25 kg/m2 (<23 kg/m2 for South Asians). CVD, cardiovascular disease; eGFR, estimated glomerular filtration rate; HDL, high density lipoprotein; NHSHC, National Health Service
Health Check. Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 8 Open access Open access
on October 23, 2024 by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from on Octobe
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BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
n: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from 9
J Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025
gnitude of changes in the individual risk factors during follow-up. BMI, body mass index; NHSHC, National Health
Check; SBP, systolic blood pressure; TCh, total cholesterol. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
ublished as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/ Figure 2 Magnitude of changes in the individual risk factors during follow-up. BMI, body mass index; NHSHC, National Health
Service Health Check; SBP, systolic blood pressure; TCh, total cholesterol. Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 9 Open access Open access factors is relevant to the severity of the disease.8 10 Based
on the example of high BP management, our study
provides strong evidence of the clinical effectiveness of
NHSHC as a public programme aimed at reduction of
CVD risk. However, the general perception of NHSC
should be changed. NHSHC has to be recognised as a
sequence of events starting with detection of increased
CVD risk during the NHSHC, followed by general advice
and prescription of therapy, and follow-up monitoring. Then a reassessment is required to decide if further
changes to therapy are needed. (OR (95% CI) (1.79 (1.33 to 2.17), p<0.001), younger
age (−1.02 (−1.06 to −1.001), p=0.004), greater SBP (1.02
(1.01 to 1.03), p<0.001), greater BMI (1.02 (1.01 to 1.04),
p=0.001) and higher Townsend score (more deprived)
(1.04 (1.01 to 1.06)), were independently associated with
presence of follow-up record. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 2023. Downloaded from REFERENCES 2013 ESH/ESC guidelines
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not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been
peer-reviewed. Any opinions or recommendations discussed are solely those
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability
of the translations (including but not limited to local regulations, clinical guidelines,
terminology, drug names and drug dosages), and is not responsible for any error
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checks on the prevalence of disease in general practices: a
controlled study. Br J Gen Pract 2014;64:e516–21. Funding The GENVASC study is funded by the NIHR Leicester Biomedical Research
Centre (grant no. BRC-1215-20010). RD is funded by NIHR (grant number NA). KK
is supported by the National Institute for Health Research (NIHR) Applied Research
Collaboration East Midlands (ARC EM) and the NIHR Leicester Biomedical Research
Centre (BRC) (grant number NA). y
10 Kennedy O, Su F, Pears R, et al. Evaluating the effectiveness of the
NHS health check programme in South England: a quasi-randomised
controlled trial. BMJ Open 2019;9:e029420. p
11 Cochrane T, Davey R, Iqbal Z, et al. Nhs health checks through
general practice: randomised trial of population cardiovascular risk
reduction. BMC Public Health 2012;12:944. Map disclaimer The inclusion of any map (including the depiction of any
boundaries therein), or of any geographic or locational reference, does not imply
the expression of any opinion whatsoever on the part of BMJ concerning the legal
status of any country, territory, jurisdiction or area or of its authorities. Any such
expression remains solely that of the relevant source and is not endorsed by BMJ. Maps are provided without any warranty of any kind, either express or implied. 2 England PH. NHS health check best practice guidance. 2 13 Hippisley-Cox J, Coupland C, Vinogradova Y, et al. Predicting
cardiovascular risk in England and Wales: prospective derivation and
validation of QRISK2. BMJ 2008;336:1475–82. 14 (NICE) NIfHaCE. Cardiovascular disease: risk assessment and
reduction, including lipid modification. clinical guideline CG181. 2014. Competing interests KK was a national advisor for the NHS Health Checks
programme. Competing interests KK was a national advisor for the NHS Health Checks
programme. 15 Centere NLBR. n.d. Available: https://www2.le.ac.uk/projects/bru/
our-research/research-themes/genetics-and-biomarkers/genvasc Patient and public involvement Patients and/or the public were involved in the
design, or conduct, or reporting, or dissemination plans of this research. Refer to
the Methods section for further details. on October 23, 2024 by guest. Protected by copyright. /bmjopen.bmj.com/ 16 Patel R, Barnard S, Thompson K, et al. Evaluation of the uptake
and delivery of the NHS health check programme in England, using
primary care data from 9.5 million people: a cross-sectional study. BMJ Open 2020;10:e042963. Patient consent for publication Not applicable. 17 Mancia G, Fagard R, Narkiewicz K, et al. REFERENCES 10Institute of Cardiovascular Science, University College London, London, UK 1 Murray CJL, Richards MA, Newton JN, et al. Uk health performance:
findings of the global burden of disease study 2010. Lancet
2013;381:997–1020. Acknowledgements The research team would like to express gratitude to all
participant of the GENVASC study. We would also like to thank all healthcare and
other professionals involved in performing NHS Health Check as well as research
related tasks. 2 Roth GA, Johnson C, Abajobir A, et al. Global, regional, and national
burden of cardiovascular diseases for 10 causes, 1990 to 2015. J Am
Coll Cardiol 2017;70:1–25. i 3 Department of Health. Putting prevention first: vascular checks risk
assessment and management - impact assessment. 2008. Contributors RD, RP, NJS: designed the project, performed analysis and are
gurantors and are responsible for the overall contents of the manuscript. VB:
designed the project, performed analysis, supervised the statistical analysis and is
responsible for the overall contents of the manuscript. DL, CG, RB, SS: took part in
collection and analysis of data, was involved in managing of the research databases
and download of data, reviewed the content and contributed to the final manuscript. EB: took part in collection and analysis of data, was involved in overall management
of the study, reviewed the content and contributed to the final manuscript. SK, AK,
AZ, AC, AH, KK, NJ, ML, AF: took part in collection and analysis of data, reviewed
the content and contributed to the final manuscript. MN: took part in collection and
analysis of data, was involved in and overlooked clinical governance of the project,
reviewed the content and contributed to the final manuscript. AM: took part in
collection and analysis of data, was involved in overall management of the study,
reviewed the content and contributed to the final manuscript. 4 Department of Health. Economic modelling for vascular checks. 2008. 5 Krogsbøll LT, Jørgensen KJ, Gøtzsche PC. Universal health checks
should be abandoned. BMJ 2013;347:bmj.f5227. 6 Soljak M, Majeed A, Millett C. Response to krogsboll and colleagues:
NHS health checks or government by randomised controlled trial? BMJ 2013;347:bmj.f5984. 7 Chang KC-M, Lee JT, Vamos EP, et al. Impact of the National health
service health check on cardiovascular disease risk: a difference-in-
differences matching analysis. CMAJ 2016;188:E228–38. 8 Forster AS, Dodhia H, Booth H, et al. Estimating the yield of NHS
health checks in England: a population-based cohort study. Radoslaw Debiec http://orcid.org/0000-0003-2292-467X
Anuj Chahal http://orcid.org/0000-0001-5664-4487
Riyaz Patel http://orcid.org/0000-0003-4603-2393 5Diabetes and Cardiovascular Medicine General Practice Alliance Federation Radoslaw Debiec http://orcid.org/0000-0003-2292-467X
Anuj Chahal http://orcid.org/0000-0001-5664-4487
Riyaz Patel http://orcid.org/0000-0003-4603-2393 Radoslaw Debiec http://orcid.org/0000-0003-2292-467X
Anuj Chahal http://orcid.org/0000-0001-5664-4487
Riyaz Patel http://orcid.org/0000-0003-4603-2393 5Diabetes and Cardiovascular Medicine General Prac Research and Training Academy, Northampton, UK Riyaz Patel http://orcid.org/0000-0003-4603-2393 6South Leicestershire Medical Group, Kibworth Beauchamp, UK 7Lakeside Healthcare Research, Corby, UK 8Willowbrook Medical Centre, Leicester, UK 9Department of Health Sciences, University of Leicester, Leicester, UK Limitations of the study Our analysis has several limitations. Not all patients
attending the NHSHC and invited to the GENVASC
study took part and the exact proportion (uptake) is not
known. It is possible that the patients who participated
in the study were more proactive in attending follow-up
appointments and undertaking medical and lifestyle
interventions than the general population of attenders to
the NHSHC. Our analysis was performed in a population
with higher than the average for UK proportion of Asian
participants. The performed analysis focused on CVD risk
factors recorded in the primary care records but no data
were available regarding lifestyle changes (eg, on phys-
ical exercise, diet or alcohol consumption). Furthermore,
we do not have data on prescription of antiobesity drugs,
or referral to weight loss, alcohol or diabetes prevention
services. 2024 by guest. Protected by copyright. uest. Protected by copyright. Author affiliations
1Department of Cardiovascular Sciences and NIHR Cardiovascular Research Centre,
University of Leicester, Leicester, UK
2Department of Food Science and Nutrition, University of the Aegean, Lemnos,
Greece
3 Our findings are consistent with previous research
indicating that attendance to NHSHC is associated with
increased detection of CVD and its risk factors and
prescription of therapy.8 10 23 It also confirms the previous
findings that the magnitude of the improvement in risk 10 Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025 Open access BMJ Open: first published as 10.1136/bmjopen-2022-068025 on 30 May 20 BMJ Open: first published as 10.1136/bmjopen-2022-0680 BMJ Open: first published as 10.1136/bm 4Diabetes Research Centre, University of Leicester, Leicester, UK 4Diabetes Research Centre, University of Leicester, Leicester, UK REFERENCES Open access This is an open access article distributed in accordance with the
Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits
others to copy, redistribute, remix, transform and build upon this work for any
purpose, provided the original work is properly cited, a link to the licence is given,
and indication of whether changes were made. See: https://creativecommons.org/
licenses/by/4.0/. (
)
25 Martin A, Saunders CL, Harte E, et al. Delivery and impact of the
NHS health check in the first 8 years: a systematic review. Br J Gen
Pract 2018;68:e449–59. 26 Newton JN, Thompson K. Nhs health check: national evaluation
findings and implications. CMAJ 2017;189:E172. 11 Debiec R, et al. BMJ Open 2023;13:e068025. doi:10.1136/bmjopen-2022-068025
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The Enzymatic Doped/Undoped Poly-Silicon Nanowire Sensor for Glucose Concentration Measurement
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sensors sensors sensors sensors Cheng-Chih Hsu 1, Wen-Kai Ho 2, Chyan-Chyi Wu 3 and Ching-Liang Dai 4,* Cheng-Chih Hsu 1, Wen-Kai Ho 2, Chyan-Chyi Wu 3 and Ching-Liang Dai 4,* 1
Department of Electro-Optical Engineering, National United University, No. 2 Lienda, Miaoli 36063, Taiwan
2
Department of Electrical Engineering, Yuan Ze University, 135, Yuan-Tung Road, Chung-Li 32003, Taiwan
3
Department of Mechanical and Electromechanical Engineering, Tamkang University,
New Taipei 25137, Taiwan
4
Department of Mechanical Engineering, National Chung Hsing University, Taichung 402, Taiwan
*
Correspondence: cldai@dragon.nchu.edu.tw; Tel.: +886-4-2284-0433 1
Department of Electro-Optical Engineering, National United University, No. 2 Lienda, Miaoli 36063, Taiwan 1
Department of Electro-Optical Engineering, National United University, No. 2 Lienda, Miaoli 36063, Taiwan
2
Department of Electrical Engineering, Yuan Ze University, 135, Yuan-Tung Road, Chung-Li 32003, Taiwan 1
Department of Electro-Optical Engineering, National United University, No. 2 Lienda, Miaoli 36063, Taiwan
2
Department of Electrical Engineering, Yuan Ze University, 135, Yuan-Tung Road, Chung-Li 32003, Taiwan
3
Department of Mechanical and Electromechanical Engineering, Tamkang University,
New Taipei 25137 Taiwan 4
Department of Mechanical Engineering, National Chung Hsing University, Taichung 402, Taiwan
*
Correspondence: cldai@dragon.nchu.edu.tw; Tel.: +886-4-2284-0433 Abstract: In this work, enzymatic doped/undoped poly-silicon nanowire sensors with different
lengths were fabricated using a top-down technique to measure glucose concentration. The sensitivity
and resolution of these sensors correlate well with the dopant property and length of nanowire. Experimental results indicate that the resolution is proportional to the nanowire length and dopant
concentration. However, the sensitivity is inversely proportional to the nanowire length. The
optimum resolution can be better than 0.02 mg/dL for a doped type sensor with length of 3.5 µm. Furthermore, the proposed sensor was demonstrated for 30 applications with similar current-time
response and showed good repeatability. Keywords: nanowire sensor; glucose concentration; n-type doping Article
The Enzymatic Doped/Undoped Poly-Silicon Nanowire Sensor
for Glucose Concentration Measurement Cheng-Chih Hsu 1, Wen-Kai Ho 2, Chyan-Chyi Wu 3 and Ching-Liang Dai 4,* Citation: Hsu, C.-C.; Ho, W.-K.; Wu,
C.-C.; Dai, C.-L. The Enzymatic
Doped/Undoped Poly-Silicon
Nanowire Sensor for Glucose
Concentration Measurement. Sensors
2023, 23, 3166. https://doi.org/
10.3390/s23063166 In 2020, the U.S. Centers for Disease Control and Prevention (CDC) reported the latest
scientific data on diabetes in the United States, indicating that about 10% (29.1 million)
of the U.S. population suffers from diabetes. In 2012, diabetes-related treatment costs in
the United States were estimated to be approximately $245 billion [1]. The International
Diabetes Federation reported that 382 million people were living with diabetes in 2013
and this number is expected to increase to 592 million by 2035 [2]. In low- and middle-
income countries, diabetes continues to be a growing health burden and will increase
significantly in the coming decades. A large number of clinical studies have shown that
controlling lower blood glucose levels can reduce the risk factors of cardiovascular diseases. Self-monitoring of blood glucose levels can be effectively performed using a commercial
glucometer. According to the guideline suggested by the U.S. National Institute of Health
(NIH), patients with type 2 diabetes typically self-test before meals, after meals, and at
bedtime [1,3]. Therefore, measuring glucose concentration with a glucometer is critical to
reducing the costs associated with diabetes. Citation: Hsu, C.-C.; Ho, W.-K.; Wu,
C.-C.; Dai, C.-L. The Enzymatic
Doped/Undoped Poly-Silicon
Nanowire Sensor for Glucose
Concentration Measurement. Sensors
2023, 23, 3166. https://doi.org/
10.3390/s23063166 Academic Editors: Antonio Di
Bartolomeo and Charlene Lobo Received: 10 February 2023
Revised: 3 March 2023
Accepted: 14 March 2023
Published: 16 March 2023 There are various methods [4–20] to measure blood glucose concentration, which can
be divided into non-invasive [4–9] and invasive [10–20]. Since the optical method [4–9]
can realize the non-invasive measurement of blood glucose concentration, it has become
a focus for technological development in recent decades. Although non-invasive blood
glucose monitoring technology has the advantage of painless monitoring of blood glucose
concentration, individual patient differences (including race, skin color, skin composition
thickness, and complex blood components) will lead to various errors in measurement
results and reduce the reliability of clinical diagnosis. Therefore, currently available meth-
ods for clinical monitoring of blood glucose levels are based on the invasive (in vitro)
methods. To avoid the influence of complex chemicals from blood, these methods applied
a specific enzyme to catalyze glucose into a unique resultant, which induced changes in the Copyright:
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/sensors Sensors 2023, 23, 3166. https://doi.org/10.3390/s23063166 Sensors 2023, 23, 3166 2 of 17 physical properties of the testing sample (such as refractive index (RI) [7], light intensity
and polarization state [8], wavelength shift [9], or electric current [10–20]). Thus, these
methods provide less individual variability and high reliability. Many studies [10–20]
pointed out the possibility of measuring glucose concentration using nanowire-structured
sensors. Nanowire sensors work on the principle that the charges around the structure
will create a surface potential on the nanowire surface, which translates into a change in
drain–source current [11,12]. Chen et al. [13] reviewed the current literature on Si nanowire
field effect transistors (FETs) for biosensing and highlighted some advantages of enzyme-
modified FETs for real-time quantitative analysis. Rahman et al. [14] comprehensively
reviewed nanostructured metal oxide glucose biosensors and highlighted the advantages
and drawbacks of enzymatic and non-enzymatic glucose sensors. The enzymatic glucose
sensor exhibits high selectivity under appropriate pH conditions. Despite the better pH
immunity of non-enzymatic sensors, the lower selectivity and rapid inactivation of such
sensors remains a priority. Shao et al. [15] developed silicon nanowires with boron modifi-
cation for glucose concentration measurement. On the surface of the boron modified silicon
nanowire, a Donnan potential occurred and varied with the glucose concentration. The
resistance of the nanowire sensor was measured to obtain the glucose concentration. The
results showed good sensitivity and around 172 nAmM−1. Shervedani et al. [16] fabricated
an enzyme-free glucose sensor using 3D spiny nickel nanowire on a copper substrate. The
novel sensor structure provided good enhancement of electrocatalytic behavior for glucose
oxidation. The sensitivity of their method could be better than 4000 µAcm−2mM−2 and
the detection limit could be down to 0.1 µM. Li et al. [17] proposed a nonenzymatic glucose
sensor with Pt-Pd nanowire arrays. Based on the electrochemical active surface area caused
by the effective channel of the hydrogen adsorption/desorption, the sensors provided the
sensitivity of 41.5 µAcm−2mM−2. Horng et al. [18] demonstrated an enzymatic glucose
sensor that incorporated glucose oxidase (GOx) and direct-growth polaniline nanowires
(PANI-NWs). The PANI-NWs enhanced redox-response resulted from the larger reaction
surface area and lower resistance. The sensitivity could be better than 2.5 mAcm−2mM−2
and the detection limit would be lower than 0.05 mM. Dawson et al. [19] employed a
redox mediator (FcCOOH) into an enzymatic gold nanowire glucose sensor. As a result
of eliminating the oxygen consumption and interference effects of the mediator, the novel
sensor amplified the electrochemical signal and therefore the detection limit could be down
to 3 µM. Previous study [20] proposed un-doping multiple silicon nanowire sensor with en-
zymatic modification for measuring glucose concentration. The sensitivity of the nanowire
sensor is inversely proportional to the length, and the sensor can take 10 consecutive
measurements with similar results. In this work, the top-down method and the technique of immobilizing glucose oxidase
on nanowires were used to prepare doped/undoped poly-silicon nanowire sensors to
measure glucose concentration. Using re-oxidation and oxide stripping procedures, the
nanowire width was trimmed to a size of 130 nm with two different nanowire lengths
(3.5 µm and 5.3 µm). The n-type (phosphorus) dopant was selected and the concentration
was controlled at 5 × 1018 cm−3. Experimental results show that the sensitivity is inversely
proportional to the length of the nanowire. The highest sensitivity of 0.008 µA/(mg/dL) can
be obtained when the nanowire length is 3.5 µm. Theoretical and actual resolutions of the
proposed sensor are 0.013 mg/dL and 1.75 mg/dL, respectively. Reliability is demonstrated
by calculating the relative standard deviation (RSD), and the average RSD of 30 replicates
for each glucose concentration can be better than 10%. Furthermore, the proposed sensor
can be reused 30 times at an acceptable performance level. 2. Fabrication Procedure for the Enzymatic Poly-Silicon Nanowire Glucose Sensor Therefore, a source/drain pad region was formed on the bottom dielectric layer with n-
type dopant (concentration of 5 × 1018 cm−3) by ion implantation. To adjust the dimensions
of poly-silicon nanowire, a 30 nm thick thermal oxidation was performed at 900 °C fol-
lowed by removal of the oxide. Finally, a poly-silicon nanowire width of about 130 nm
could be reduced after the re-oxidation and oxide lift-off process. According to the previ-
ous study, short nanowire length exhibited high resolution [21]. Therefore, the length of
the nanowires was controlled to around 3 μm to 5 μm in this study. The n-type (phospho-
rus) dopant was implanted and concentration was controlled at 5 × 1018 cm−3. method [13,16] and the silicon nanowire features are completed using nanopatterned I-line
lithography. A 35 nm thick oxide layer was grown using thermal oxidation at 900 ◦C,
followed by 30 nm thick Si3N4 deposited using low pressure chemical vapor deposition
method (LPCVD) at 780 ◦C. Next, a layer about 2 nm thick was grown on the silicon nitride
as the bottom dielectric layer using thermal oxidation at 900 ◦C. A 60 nm thick poly-silicon
layer was deposited for the channel using LPCVD at 550 ◦C. Thereafter, I-line lithography
and the photoresistor trimming process were performed together, followed by Si etching. Therefore, a source/drain pad region was formed on the bottom dielectric layer with n-type
dopant (concentration of 5 × 1018 cm−3) by ion implantation. To adjust the dimensions of
poly-silicon nanowire, a 30 nm thick thermal oxidation was performed at 900 ◦C followed
by removal of the oxide. Finally, a poly-silicon nanowire width of about 130 nm could be
reduced after the re-oxidation and oxide lift-off process. According to the previous study,
short nanowire length exhibited high resolution [21]. Therefore, the length of the nanowires
was controlled to around 3 µm to 5 µm in this study. The n-type (phosphorus) dopant was
implanted and concentration was controlled at 5 × 1018 cm−3. tion conditions. The proposed nanowire sensor was fabricated using a top down method
[13,16] and the silicon nanowire features are completed using nanopatterned I-line lithog-
raphy. A 35 nm thick oxide layer was grown using thermal oxidation at 900 °C, followed
by 30 nm thick Si3N4 deposited using low pressure chemical vapor deposition method
(LPCVD) at 780 °C. 2. Fabrication Procedure for the Enzymatic Poly-Silicon Nanowire Glucose Sensor Figure 1 shows the fabrication process for the proposed nanowire glucose sensor
(NWGS) with nanowires of different length, and the details are described below. In
this work, two lengths of doped/undoped nanowires were fabricated under the same
fabrication conditions. The proposed nanowire sensor was fabricated using a top-down Sensors 2023, 23, 3166 3 of 17
sensor
In this 3 of 17
sensor
In this method [13,16] and the silicon nanowire features are completed using nanopatterned I-line
lithography. A 35 nm thick oxide layer was grown using thermal oxidation at 900 ◦C,
followed by 30 nm thick Si3N4 deposited using low pressure chemical vapor deposition
method (LPCVD) at 780 ◦C. Next, a layer about 2 nm thick was grown on the silicon nitride
as the bottom dielectric layer using thermal oxidation at 900 ◦C. A 60 nm thick poly-silicon
layer was deposited for the channel using LPCVD at 550 ◦C. Thereafter, I-line lithography
and the photoresistor trimming process were performed together, followed by Si etching. Therefore, a source/drain pad region was formed on the bottom dielectric layer with n-type
dopant (concentration of 5 × 1018 cm−3) by ion implantation. To adjust the dimensions of
poly-silicon nanowire, a 30 nm thick thermal oxidation was performed at 900 ◦C followed
by removal of the oxide. Finally, a poly-silicon nanowire width of about 130 nm could be
reduced after the re-oxidation and oxide lift-off process. According to the previous study,
short nanowire length exhibited high resolution [21]. Therefore, the length of the nanowires
was controlled to around 3 µm to 5 µm in this study. The n-type (phosphorus) dopant was
implanted and concentration was controlled at 5 × 1018 cm−3. tion conditions. The proposed nanowire sensor was fabricated using a top down method
[13,16] and the silicon nanowire features are completed using nanopatterned I-line lithog-
raphy. A 35 nm thick oxide layer was grown using thermal oxidation at 900 °C, followed
by 30 nm thick Si3N4 deposited using low pressure chemical vapor deposition method
(LPCVD) at 780 °C. Next, a layer about 2 nm thick was grown on the silicon nitride as the
bottom dielectric layer using thermal oxidation at 900 °C. A 60 nm thick poly-silicon layer
was deposited for the channel using LPCVD at 550 °C. Thereafter, I-line lithography and
the photoresistor trimming process were performed together, followed by Si etching. 2. Fabrication Procedure for the Enzymatic Poly-Silicon Nanowire Glucose Sensor Next, a layer about 2 nm thick was grown on the silicon nitride as the
bottom dielectric layer using thermal oxidation at 900 °C. A 60 nm thick poly-silicon layer
was deposited for the channel using LPCVD at 550 °C. Thereafter, I-line lithography and
the photoresistor trimming process were performed together, followed by Si etching. Therefore, a source/drain pad region was formed on the bottom dielectric layer with n-
type dopant (concentration of 5 × 1018 cm−3) by ion implantation. To adjust the dimensions
of poly-silicon nanowire, a 30 nm thick thermal oxidation was performed at 900 °C fol-
lowed by removal of the oxide. Finally, a poly-silicon nanowire width of about 130 nm
could be reduced after the re-oxidation and oxide lift-off process. According to the previ-
ous study, short nanowire length exhibited high resolution [21]. Therefore, the length of
the nanowires was controlled to around 3 μm to 5 μm in this study. The n-type (phospho-
rus) dopant was implanted and concentration was controlled at 5 × 1018 cm−3. Figure 1. Schematic of the fabrication procedures of NWGS. Figure 1. Schematic of the fabrication procedures of NWGS. Figure 1. Schematic of the fabrication procedures of NWGS. Figure 1. Schematic of the fabrication procedures of NWGS. Figure 1. Schematic of the fabrication procedures of NWGS. Figure 1. Schematic of the fabrication procedures of NWGS. Figure 1. Schematic of the fabrication procedures of NWGS. Figure 1. Schematic of the fabrication procedures of NWGS. After fabricating the nanowire devices, the devices were covered with a 10% (v/v)
ethanol solution of 3-aminopropyltriethoxysilane (APTES) for 15 min at room temper-
ature. The solution was then removed and baked at 100 ◦C for 45 min to modify the
nanowire’s surface. To covalently immobilize GOx on SiO2, 0.01% suberic acid bis(3-sulfo-
N-hydroxysuccinimide ester) sodium salt (BS3) was mixed in 10 mM phosphate buffered Sensors 2023, 23, 3166 4 of 17
mpera-
he nan- 4 of 17
mpera-
he nan- saline (PBS) solution, and the mixture covered the nanowire surface for 30 min at room
temperature. The modified surface was covered with a 150 µg/mL GOx solution at pH 7
for 1 h. Unreacted aldehyde groups were quenched by immersion in 15 mM Tris buffer
solution for 15 min at room temperature [20]. 2. Fabrication Procedure for the Enzymatic Poly-Silicon Nanowire Glucose Sensor Figure 2a shows the photographs of the
NWGS with doped/undoped process and the top-view SEM images of NWGS with various
lengths are shown in Figure 2b,c. It is clear that the length of the nanowire is well controlled
at around 3 µm and 5 µm. hydroxysuccinimide ester) sodium salt (BS3) was mixed in 10 mM phosphate buffered
saline (PBS) solution, and the mixture covered the nanowire surface for 30 min at room
temperature. The modified surface was covered with a 150 µg/mL GOx solution at pH 7 for
1 h. Unreacted aldehyde groups were quenched by immersion in 15 mM Tris buffer solution
for 15 min at room temperature [20]. Figure 2a shows the photographs of the NWGS with
doped/undoped process and the top-view SEM images of NWGS with various lengths are
shown in Figure 2b,c. It is clear that the length of the nanowire is well controlled at around
3 μm and 5 μm. Figure 2. Photographs and top-view SEM images of NWGS. (a) photographs of the NWGS; (b) top-
view SEM image of doped/undoped nanowire sensor with length of 3.5 μm; (c) top-view SEM image
of doped/undoped nanowire sensor with length of 5.3 μm. 3 Th
ti
l Si
l ti
Figure 2. Photographs and top-view SEM images of NWGS. (a) photographs of the NWGS; (b) top-
view SEM image of doped/undoped nanowire sensor with length of 3.5 µm; (c) top-view SEM image
of doped/undoped nanowire sensor with length of 5.3 µm. h
l
l Figure 2. Photographs and top-view SEM images of NWGS. (a) photographs of the NWGS; (b) top-
view SEM image of doped/undoped nanowire sensor with length of 3.5 μm; (c) top-view SEM image
of doped/undoped nanowire sensor with length of 5.3 μm. Figure 2. Photographs and top-view SEM images of NWGS. (a) photographs of the NWGS; (b) top-
view SEM image of doped/undoped nanowire sensor with length of 3.5 µm; (c) top-view SEM image
of doped/undoped nanowire sensor with length of 5.3 µm. 3. Theoretical Simulation
3. Theoretical Simulation Higher dopant concentration of the nanowire implies a larger variation of drain current. Figure 3. Simulation results of Id-Vg curve for various dopant concentrations of the poly-silicon nan-
owire. Figure 3. Simulation results of Id-Vg curve for various dopant concentrations of the poly-silicon
nanowire. gure 3. Simulation results of Id-Vg curve for various dopant concentrations of the poly-silicon nan-
wire. Figure 3. Simulation results of Id-Vg curve for various dopant concentrations of the poly-silicon
nanowire. Figure 4a summarizes the Id-Vg curve measurement results of doped/undoped
NWGS with the lengths of 3.5 μm (for A and C) and 5.3 μm (for B and D), where labels A
nd B indicate the doped type NWGS, and labels C and D represent the undoped type
NWGS. The dopant concentration of NWGS was controlled at 5 × 1018 cm−3. It is clear that
he variation of Id increases with increasing Vg, and the threshold voltage Vth can be deter-
mined by the intercept of the Vg-axis in the Id-Vg curve [11,12]. The threshold voltages (Vth)
f those NWGS are approximately 1.3 V (curve A), 1.25 V (curve B), 1.43 V (curve C), and
.67 V (curve D), respectively. The results show that Vth decreases with the increase in
anowire dopant concentration. Figure 4b shows that the Id-Vd curves of the doped/un-
oped NWGS with the gate voltage controlled at 2 V. The results show the Vd should be
within 0.75 V to maintain the device operation in a linear range. The results show that the
oped NWGS with a shorter length has a more sensitive Id-Vd response, and its response
evel is about 2 times that of the doped NWGS with longer length and 6 times that of the
Figure 4a summarizes the Id-Vg curve measurement results of doped/undoped NWGS
with the lengths of 3.5 µm (for A and C) and 5.3 µm (for B and D), where labels A and B
indicate the doped type NWGS, and labels C and D represent the undoped type NWGS. The
dopant concentration of NWGS was controlled at 5 × 1018 cm−3. It is clear that the variation
of Id increases with increasing Vg, and the threshold voltage Vth can be determined by the
intercept of the Vg-axis in the Id-Vg curve [11,12]. 3. Theoretical Simulation
3. Theoretical Simulation The electrical behavior of nanowire can be described using the Id-Vg and Id-Vd curves. Different physical dimensions and dopant properties of the nanowires lead to different Id-
Vg curve behaviors, indicating that the appropriate geometry and dopant concentration
of the nanowires exhibited high sensitivity for biosensing purposes. Eflstrom et al. [12]
The electrical behavior of nanowire can be described using the Id-Vg and Id-Vd curves. Different physical dimensions and dopant properties of the nanowires lead to different
Id-Vg curve behaviors, indicating that the appropriate geometry and dopant concentration
of the nanowires exhibited high sensitivity for biosensing purposes. Eflstrom et al. [12]
indicated that the device sensitivity increased with decreased nanowire width. Previous
work [20] reported that the threshold voltage and resolution of nanowires increased with
increasing nanowire length. Cui et al. [22] showed that the resistivity of n-type nanowires
exhibited more than 4 orders of magnitude smaller than the undoped nanowires. They also Sensors 2023, 23, 3166 5 of 17
ires
hey 5 of 17
ires
hey pointed out the n-type nanowire exhibited lower Fermi level when operated at the Vg > 0
condition, in which electrons will accumulate in nanowire and enhanced conductivity. These enhancements might be increasing the sensitivity of nanowire. This work simulates
the Id-Vg curve of the nanowire under various dopant concentrations using commercial
software (Synopsys, TCAD) by setting of the nanowire length and width to be 3 µm and
150 nm, respectively. The drain voltage was 0.5 V. Figure 3a indicates that the Id-Vg curve of
various dopant concentrations of the nanowire, in which the curves (i) to (iii) corresponded
to dopant concentration 1 × 1018 cm−3, 1 × 1017 cm−3, and undoped, respectively. Higher
dopant concentration of the nanowire implies a larger variation of drain current. 0 condition, in which electrons will accumulate in nanowire and enhanced conductivity. hese enhancements might be increasing the sensitivity of nanowire. This work simulates
he Id-Vg curve of the nanowire under various dopant concentrations using commercial
oftware (Synopsys, TCAD) by setting of the nanowire length and width to be 3 μm and
50 nm, respectively. The drain voltage was 0.5 V. Figure 3a indicates that the Id-Vg curve
f various dopant concentrations of the nanowire, in which the curves (i) to (iii) corre-
ponded to dopant concentration 1 1018 cm−3, 1 1017 cm−3, and undoped, respectively. 3. Theoretical Simulation
3. Theoretical Simulation The threshold voltages (Vth) of those
NWGS are approximately 1.3 V (curve A), 1.25 V (curve B), 1.43 V (curve C), and 1.67 V
(curve D), respectively. The results show that Vth decreases with the increase in nanowire
dopant concentration. Figure 4b shows that the Id-Vd curves of the doped/undoped NWGS
with the gate voltage controlled at 2 V. The results show the Vd should be within 0.75 V to
maintain the device operation in a linear range. The results show that the doped NWGS
with a shorter length has a more sensitive Id-Vd response, and its response level is about
2 times that of the doped NWGS with longer length and 6 times that of the undoped NWGS. 6 of 17 Sensors 2023, 23, 3166 Figure 4. Electrical characteristic curves of NWGS with lengths of 3.5 μm and 5.3 μm. (a) Id-Vg meas-
urement for doped/undoped poly-silicon nanowire with different nanowire length; (b) Id-Vd meas-
urement for doped/undoped poly-silicon nanowire with different nanowire length. Figure 4. Electrical characteristic curves of NWGS with lengths of 3.5 µm and 5.3 µm. (a) Id-Vg
measurement for doped/undoped poly-silicon nanowire with different nanowire length; (b) Id-Vd
measurement for doped/undoped poly-silicon nanowire with different nanowire length. 4. Experimental Results
Figure 5a shows a measurement setup similar to Bergveld’s method [23] and Figure 5b
shows an actual photo of the proposed system. Performance of the proposed poly-silicon
nanowire sensor was demonstrated by measuring various glucose concentrations prepared
b
dissol ing different weights of glucose in DI water The test sample with a
olume of Figure 4. Electrical characteristic curves of NWGS with lengths of 3.5 μm and 5.3 μm. (a) Id-Vg meas-
t f
d
d/
d
d
l
ili
i
ith diff
t
i
l
th (b) I V
Figure 4. Electrical characteristic curves of NWGS with lengths of 3.5 µm and 5.3 µm. (a) Id-Vg
measurement for doped/undoped poly-silicon nanowire with different nanowire length; (b) Id-Vd
measurement for doped/undoped poly-silicon nanowire with different nanowire length. gure 4. Electrical characteristic curves of NWGS with lengths of 3.5 μm and 5.3 μm. (a) Id-Vg meas-
Figure 4. Electrical characteristic curves of NWGS with lengths of 3.5 µm and 5.3 µm. (a) Id-Vg
measurement for doped/undoped poly-silicon nanowire with different nanowire length; (b) Id-Vd
measurement for doped/undoped poly-silicon nanowire with different nanowire length. ement for doped/undoped po
ement for doped/undoped po
4. Experimental Results ement for doped/undoped poly-silicon nanowire with different nanowire length. Figure 5a shows a measurement setup similar to Bergveld’s method [23] and Figure 5b
shows an actual photo of the proposed system. Performance of the proposed poly-silicon
nanowire sensor was demonstrated by measuring various glucose concentrations prepared
by dissolving different weights of glucose in DI water. The test sample with a volume of 7 of 17
oly sili
ns pre- Sensors 2023, 23, 3166 1 cc was injected from the injection port of the microfluidic channel and reacted with the
NWGS for 10 s. Next, the reacted liquid was sucked out through the microfluidic channel’s
out port while 10 cc of DI water was injected from the injection port for NWGS cleaning. The current meter (Agilent U2722A) has a resolution of 1 nA in the ±10 µA current range. In this study, the Vg and Vd were controlled at 2 V and 0.5 V, respectively. This study will
verify the repeatability test and reliability verification of the NWGS, respectively. j
j
p
with the NWGS for 10 s. Next, the reacted liquid was sucked out through the microfluidic
channel’s out port while 10 cc of DI water was injected from the injection port for NWGS
cleaning. The current meter (Agilent U2722A) has a resolution of 1 nA in the ±10 μA cur-
rent range. In this study, the Vg and Vd were controlled at 2 V and 0.5 V, respectively. This
study will verify the repeatability test and reliability verification of the NWGS, respec-
tively. Figure 5. The measurement system of proposed method. (a) Diagram of the experimental setup; (b)
real picture of the measurement system. Figure 5. The measurement system of proposed method. (a) Diagram of the experimental setup;
(b) real picture of the measurement system. Figure 5. The measurement system of proposed method. (a) Diagram of the experimental setup; (b)
real picture of the measurement system. Figure 5. The measurement system of proposed method. (a) Diagram of the experimental setup;
(b) real picture of the measurement system. To validate the NWGS repeatability test, glucose concentrations were varied from 10
mg/dL to 300 mg/dL and measured using the NWGS. Figure 6a,b shows the current-time
response curve of the NWGS during the continuous additions of glucose solution. Figure
6a indicates that the drain current increases with an increasing concentration of glucose. ement for doped/undoped po
ement for doped/undoped po
4. Experimental Results It is clear that the current-time response curves of the doped type NWGS increase signifi-
To validate the NWGS repeatability test, glucose concentrations were varied from
10 mg/dL to 300 mg/dL and measured using the NWGS. Figure 6a,b shows the current-
time response curve of the NWGS during the continuous additions of glucose solution. Figure 6a indicates that the drain current increases with an increasing concentration of
glucose. It is clear that the current-time response curves of the doped type NWGS increase
significantly as the glucose solution concentration is lower than 10 mg/dL. On the con-
trary, undoped type NWGS have no obvious change in drain current until the glucose
concentration is greater than 50 mg/dL. The reason might derive from the dopant nanowire
sensor reducing the resistivity [22] of the nanowire and lead to the greater sensitivity of the
current-time response of nanowire sensor. 8 of 17
sensor
he cur- Sensors 2023, 23, 3166 Figure 6. Current-time response curve of doped/undoped poly-Silicon nanowire wit
owire length. (a) Relationship between the drain current and concentration of glucos
bility of the doped/undoped nanowire sensor with length of 3.5 μm. Figure 6. Current-time response curve of doped/undoped poly-Silicon nanowire
nanowire length. (a) Relationship between the drain current and concentration of g
peatability of the doped/undoped nanowire sensor with length of 3.5 µm. The repeatability of the proposed sensor was verified by performing 30
measurements on a glucose concentration of 300 mg/dL, where sensors A
selected for comparison, as shown in Figure 6b. The average values of Id of
C were 2.41 µA and 0.45 µA, respectively. Moreover, the standard deviati
corresponding to each sensor was 0 06 and 0 14 respectively Clearly Figure 6 Figure 6. Current-time response curve of doped/undoped poly-Silicon nanowire with various nan-
owire length. (a) Relationship between the drain current and concentration of glucose; (b) Repeata-
bility of the doped/undoped nanowire sensor with length of 3.5 μm. Figure 6. Current-time response curve of doped/undoped poly-Silicon nanowire with various
nanowire length. (a) Relationship between the drain current and concentration of glucose; (b) Re-
peatability of the doped/undoped nanowire sensor with length of 3.5 µm. The repeatability of the proposed sensor was verified by performing 30 consecutive
measurements on a glucose concentration of 300 mg/dL, where sensors A and C were
selected for comparison, as shown in Figure 6b. The average values of Id of sensor A and Figure 6. ement for doped/undoped po
ement for doped/undoped po
4. Experimental Results Current-time response curve of doped/undoped poly-Silicon nanowire with various nan-
owire length. (a) Relationship between the drain current and concentration of glucose; (b) Repeata-
bility of the doped/undoped nanowire sensor with length of 3.5 μm. Figure 6. Current-time response curve of doped/undoped poly-Silicon nanowire with various
nanowire length. (a) Relationship between the drain current and concentration of glucose; (b) Re-
peatability of the doped/undoped nanowire sensor with length of 3.5 µm. The repeatability of the proposed sensor was verified by performing 30 consecutive
measurements on a glucose concentration of 300 mg/dL, where sensors A and C were
selected for comparison, as shown in Figure 6b. The average values of Id of sensor A and
C were 2.41 µA and 0.45 µA, respectively. Moreover, the standard deviation (SD) of Id
corresponding to each sensor was 0.06 and 0.14, respectively. Clearly, Figure 6b shows that Sensors 2023, 23, 3166 9 of 17
Id
ws 9 of 17
Id
ws the current-time response curves exhibit high similarity in 30 consecutive applications of
each sensor. ns of each sensor. Figure 7 shows the smallest glucose concentration measurement with sensor A. The
ults show that the difference of Id between 5 mg/dL and 2 mg/dL is about 50 nA Con Figure 7 shows the smallest glucose concentration measurement with sensor A. The
results show that the difference of Id between 5 mg/dL and 2 mg/dL is about 50 nA. Considering the resolution of the current meter (0.1 nA), sensor A should theoretically be
able to measure a change of 1 mg/dL. Therefore, the proposed sensor with short nanowire
length and n-type doping has a high possibility of measuring the glucose concentration
below 1 mg/dL. ults show that the difference of Id between 5 mg/dL and 2 mg/dL is about 50 nA. Con-
ering the resolution of the current meter (0.1 nA), sensor A should theoretically be able
measure a change of 1 mg/dL. Therefore, the proposed sensor with short nanowire
gth and n-type doping has a high possibility of measuring the glucose concentration
ow 1 mg/dL. ure 7. Current-time response curve for smallest glucose concentration measurement using a
ped nanowire sensor with a length of 3.5 μm. Figure 7. Current-time response curve for smallest glucose concentration measurement using a
doped nanowire sensor with a length of 3.5 µm. ure 7. ement for doped/undoped po
ement for doped/undoped po
4. Experimental Results Current-time response curve for smallest glucose concentration measurement using a
ed nanowire sensor with a length of 3.5 μm. Figure 7. Current-time response curve for smallest glucose concentration measurement using a
doped nanowire sensor with a length of 3.5 µm. The reliability evaluation was performed by calculating the relative standard devia-
n (RSD) of 30 replicate experiments for each glucose concentration in accordance with
rke’s method [24] The RSD can be expressed as
The reliability evaluation was performed by calculating the relative standard deviation
(RSD) of 30 replicate experiments for each glucose concentration in accordance with Clarke’s
method [24]. The RSD can be expressed as p
x
SD
100
RSD
=
(1)
RSD = 100 × SD
x
(1) (1) x
ere SD is standard deviation and x is the average value of the measured data. Using
ped/undoped NWGS and a traceable refractometer (reference method, model: excel-
ce R4, Mettler) to conduct 30 groups of measurements, the evaluation results are shown
Figures 8 and 9. The values in the region between the red and blue lines indicate the
asurement result within 20% of the reference concentration. The average RSD of the
ped type NWGS with lengths of 3.5 μm and 5.3 μm are 4% and 5%, respectively. The
erage RSDs for undoped NWGS are 9% and 10%, respectively. These results demon-
ate good reliability of the proposed method for both types of NWGS and satisfied the
nimum requirement (±15%) for point-of-care devices provided by the Food and Drug
where SD is standard deviation and x is the average value of the measured data. Using
doped/undoped NWGS and a traceable refractometer (reference method, model: excel-
lence R4, Mettler) to conduct 30 groups of measurements, the evaluation results are shown
in Figures 8 and 9. The values in the region between the red and blue lines indicate the
measurement result within 20% of the reference concentration. The average RSD of the
doped type NWGS with lengths of 3.5 µm and 5.3 µm are 4% and 5%, respectively. The
average RSDs for undoped NWGS are 9% and 10%, respectively. These results demon-
strate good reliability of the proposed method for both types of NWGS and satisfied the
minimum requirement (±15%) for point-of-care devices provided by the Food and Drug
Administration (FDA) [25]. x
ere SD is standard deviation and x is the average value of the measured data. ement for doped/undoped po
ement for doped/undoped po
4. Experimental Results Using
ped/undoped NWGS and a traceable refractometer (reference method, model: excel-
ce R4, Mettler) to conduct 30 groups of measurements, the evaluation results are shown
Figures 8 and 9. The values in the region between the red and blue lines indicate the
asurement result within 20% of the reference concentration. The average RSD of the
ped type NWGS with lengths of 3.5 μm and 5.3 μm are 4% and 5%, respectively. The
rage RSDs for undoped NWGS are 9% and 10%, respectively. These results demon-
ate good reliability of the proposed method for both types of NWGS and satisfied the
nimum requirement (±15%) for point-of-care devices provided by the Food and Drug
where SD is standard deviation and x is the average value of the measured data. Using
doped/undoped NWGS and a traceable refractometer (reference method, model: excel-
lence R4, Mettler) to conduct 30 groups of measurements, the evaluation results are shown
in Figures 8 and 9. The values in the region between the red and blue lines indicate the
measurement result within 20% of the reference concentration. The average RSD of the
doped type NWGS with lengths of 3.5 µm and 5.3 µm are 4% and 5%, respectively. The
average RSDs for undoped NWGS are 9% and 10%, respectively. These results demon-
strate good reliability of the proposed method for both types of NWGS and satisfied the
minimum requirement (±15%) for point-of-care devices provided by the Food and Drug
Administration (FDA) [25]. Sensors 2023, 23, 3166 10 of 17 Figure 8. The reliability evaluation of the proposed method with different length of the doped ty
NWGS. (a) nanowire lengths of 3.5 μm; (b) nanowire lengths of 5.3 μm. Figure 8. The reliability evaluation of the proposed method with different length of the doped
NWGS. (a) nanowire lengths of 3.5 µm; (b) nanowire lengths of 5.3 µm. Figure 8. The reliability evaluation of the proposed method with different length of the doped type
NWGS (a) nanowire lengths of 3 5 μm; (b) nanowire lengths of 5 3 μm
Figure 8. The reliability evaluation of the proposed method with different length of the doped type
NWGS. (a) nanowire lengths of 3.5 µm; (b) nanowire lengths of 5.3 µm. igure 8. The reliability evaluation of the proposed method with different length of the doped type
Figure 8. The reliability evaluation of the proposed method with different length of the doped type
NWGS. 5. Discussion
5. Discussion Figure 10 indicates the resulting calibration curve measured using the doped/undoped
nanowire sensor. The symbols □, #, and I represent the average value of 10 measured data
sets and the standard deviation of each concentration measured using the proposed sensor
with doped/undoped nanowire sensors, respectively. Figure 10a shows the results of the
measurements of doped nanowire sensors with nanowire of various lengths (labeled A
and B with lengths of 3.5 µm and 5.3 µm, respectively). Figure 10b shows the results of the
measurements of undoped nanowire sensors with nanowire of various lengths (labeled
C and D with lengths of 3.5 µm and 5.3 µm, respectively). The slope of the calibration
curve indicates the sensitivity (change in current per unit concentration change) of the
proposed sensor and the fact that the nanowires with shorter lengths from the device with
n-type dopant exhibited higher sensitivity. This finding correlates with the Id-Vg curves in
Figure 4. The shaper slope indicates a higher sensitivity. Figure 10 indicates the resulting calibration curve measured using the doped/un-
doped nanowire sensor. The symbols , , and I represent the average value of 10 meas-
ured data sets and the standard deviation of each concentration measured using the pro-
posed sensor with doped/undoped nanowire sensors, respectively. Figure 10a shows the
results of the measurements of doped nanowire sensors with nanowire of various lengths
(labeled A and B with lengths of 3.5 μm and 5.3 μm, respectively). Figure 10b shows the
results of the measurements of undoped nanowire sensors with nanowire of various
lengths (labeled C and D with lengths of 3.5 μm and 5.3 μm, respectively). The slope of
the calibration curve indicates the sensitivity (change in current per unit concentration
change) of the proposed sensor and the fact that the nanowires with shorter lengths from
the device with n-type dopant exhibited higher sensitivity. This finding correlates with
the Id-Vg curves in Figure 4. The shaper slope indicates a higher sensitivity. Figure 10. The calibration curves of the salt concentration measurement of the doped/undoped poly-
silicon nanowire sensors. (a) doped type NWGS with lengths of 3.5 μm (A) and 5.3 μm (B); (b)
undoped type NWGS with lengths of 3.5 μm (C) and 5.3 μm (D). Re olutio
of the p opo ed
ethod a
be ep e e ted a [26]
Figure 10. The calibration curves of the salt concentration measurement of the doped/undoped
poly-silicon nanowire sensors. ement for doped/undoped po
ement for doped/undoped po
4. Experimental Results (a) nanowire lengths of 3.5 µm; (b) nanowire lengths of 5.3 µm. 11 of 17 11 of 17 Sensors 2023, 23, 3166
,
, Figure 9 The reliability evaluation of the proposed method with different length of the undop
Figure 9. The reliability evaluation of the proposed method with different length of the u
type NWGS. (a) nanowire lengths of 3.5 µm; (b) nanowire lengths of 5.3 µm. gure 9. The reliability evaluation of the proposed method with different length of the undoped
Figure 9. The reliability evaluation of the proposed method with different length of the undoped
type NWGS. (a) nanowire lengths of 3.5 µm; (b) nanowire lengths of 5.3 µm. ure 9 The reliability evaluation of the proposed method with different length of the undoped
Figure 9. The reliability evaluation of the proposed method with different length of the undoped
type NWGS. (a) nanowire lengths of 3.5 µm; (b) nanowire lengths of 5.3 µm. Sensors 2023, 23, 3166
Sensors 2023, 23, x FOR 12 of 17
12 of 17 Resolution of the proposed method can be represented as [26]
S
I
Cres
= Resolution of the proposed method can be represented as [26]
S
I
Cres
= Resolution of the proposed method can be represented as [26]
S
I
Cres
= Cres = ∆I
S
(2)
S
ve; and ∆I and Cres represent the resolution of the
tration respectively Resolution of the measured Cres = ∆I
S
ve; and ∆I and
tration respe (2)
e where S is the slope of the calibration curve; and ∆I and Cres represent the resolution
of the current measurement and glucose concentration, respectively. Resolution of the
measured current depends on the current meter. The resolution of the current meter is
0.1 nA which can be considered as the theoretical resolution of the proposed method. There are many issues that can reduce the resolution of amperometric measurement, such
as the temperature variation, conductivity fluctuations of the electrodes, and unwanted
electrical noise from the measurement equipment. To evaluate the practical resolution
of the measured current of the proposed system, the current fluctuation of the proposed
system can be considered by applying a constant Vg to the DI water sample. In this work,
the current fluctuation is approximately 14 nA within 25 s and the results are shown in
Figure 11. According to Equation (2), the resolution of these sensors can be calculated and
are shown in Table 1. Clearly, doped NWGS exhibited higher resolution than undoped
NWGS, and the resolution of NWGS with shorter nanowire length was higher than that of
NWGS with longer nanowire length. An n-type dopant sensor with a nanowire length of
3.5 µm can achieve the optimum resolution for the proposed sensor which is approximated
of 1.75 mg/dL. rrent measurement and glucose concentration, respectively. Resolution of the measured
rrent depends on the current meter. The resolution of the current meter is 0.1 nA which
n be considered as the theoretical resolution of the proposed method. There are many
ues that can reduce the resolution of amperometric measurement, such as the temper-
ure variation, conductivity fluctuations of the electrodes, and unwanted electrical noise
om the measurement equipment. To evaluate the practical resolution of the measured
rrent of the proposed system, the current fluctuation of the proposed system can be
nsidered by applying a constant Vg to the DI water sample. In this work, the current
uctuation is approximately 14 nA within 25 s and the results are shown in Figure 11. 5. Discussion
5. Discussion (a) doped type NWGS with lengths of 3.5 µm (A) and 5.3 µm (B);
(b) undoped type NWGS with lengths of 3.5 µm (C) and 5.3 µm (D). Figure 10. The calibration curves of the salt concentration measurement of the doped/undoped poly-
silicon nanowire sensors. (a) doped type NWGS with lengths of 3.5 μm (A) and 5.3 μm (B); (b)
undoped type NWGS with lengths of 3.5 μm (C) and 5.3 μm (D). Figure 10. The calibration curves of the salt concentration measurement of the doped/undoped
poly-silicon nanowire sensors. (a) doped type NWGS with lengths of 3.5 µm (A) and 5.3 µm (B);
(b) undoped type NWGS with lengths of 3.5 µm (C) and 5.3 µm (D). Sensors 2023, 23, 3166 13 of 17 13 of 17 Resolution of the proposed method can be represented as [26]
S
I
Cres
= ccording to Equation (2), the resolution of these sensors can be calculated and are shown
Table 1. Clearly, doped NWGS exhibited higher resolution than undoped NWGS, and
e resolution of NWGS with shorter nanowire length was higher than that of NWGS with
nger nanowire length. An n-type dopant sensor with a nanowire length of 3.5 μm can
hieve the optimum resolution for the proposed sensor which is approximated of 1.75
g/dL. where S is the slope of the calibration curve; and ∆I and Cres represent the resolution
of the current measurement and glucose concentration, respectively. Resolution of the
measured current depends on the current meter. The resolution of the current meter is
0.1 nA which can be considered as the theoretical resolution of the proposed method. There are many issues that can reduce the resolution of amperometric measurement, such
as the temperature variation, conductivity fluctuations of the electrodes, and unwanted
electrical noise from the measurement equipment. To evaluate the practical resolution
of the measured current of the proposed system, the current fluctuation of the proposed
system can be considered by applying a constant Vg to the DI water sample. In this work,
the current fluctuation is approximately 14 nA within 25 s and the results are shown in
Figure 11. According to Equation (2), the resolution of these sensors can be calculated and
are shown in Table 1. Clearly, doped NWGS exhibited higher resolution than undoped
NWGS, and the resolution of NWGS with shorter nanowire length was higher than that of
NWGS with longer nanowire length. An n-type dopant sensor with a nanowire length of
3.5 µm can achieve the optimum resolution for the proposed sensor which is approximated
of 1.75 mg/dL. rrent measurement and glucose concentration, respectively. Resolution of the measured
rrent depends on the current meter. The resolution of the current meter is 0.1 nA which
n be considered as the theoretical resolution of the proposed method. There are many
ues that can reduce the resolution of amperometric measurement, such as the temper-
ure variation, conductivity fluctuations of the electrodes, and unwanted electrical noise
om the measurement equipment. To evaluate the practical resolution of the measured
rrent of the proposed system, the current fluctuation of the proposed system can be
nsidered by applying a constant Vg to the DI water sample. Resolution of the proposed method can be represented as [26]
S
I
Cres
= In this study, when n-type nanowires operate at Vd of 0.5 V,
Vth decreases as the nanowire length increases. Under the same operation, the Vth of the
undoped nanowires shows the opposite behavior. Furthermore, at the same length of
nanowires operating at Vd of 0.5 V, Vth increases with increasing doping concentration. However, when drain voltage was 1 V, the change in Vth has no obvious relationship with
the length of the nanowire. In this study, when n-type nanowires operate at Vd of 0.5 V,
Vth decreases as the nanowire length increases. Under the same operation, the Vth of the
undoped nanowires shows the opposite behavior. Furthermore, at the same length of
nanowires operating at Vd of 0.5 V, Vth increases with increasing doping concentration. p
g
g
p
g
The sensitivity of the nanowire sensor will depend on the doping concentration of
nanowire and will also depend on the evaluation method. Nair et al. [28] evaluated the
sensitivity using relative change in conductance at low drain bias and they concluded
that sensitivity was inverse to the doping concentration. Li et al. [29] followed the con-
cept and evaluated using normalized current method at Vd of 0.01 V. In contrast, this
study directly measured the drain current at various concentrations and sensitivity was
defined as the slope of the calibration curve of drain current versus concentration curve. The Vd is 20 times higher than for the Li [29] method and may induce unequal amounts
of depleted/accumulated charge or molecule conjugation on surfaces with different sur-
face densities. Therefore, that may be a possible reason why the sensitivity differs from
other methods. Figure 12 shows the SEM image of the top view of the sensor after 40 applications. Because the enzyme sensing layer catalyzes glucose and converts it to gluconic acid and
hydrogen peroxide even after each reaction, the elements are cleaned with DI water. Im-
proper cleaning methods still cannot ensure that the residual gluconic acid is completely
removed. Therefore, gluconic acid is taken up by the device and deposited around the
source, drain, and nanowire regions (marked in a red circle). Compared with previous
work [20], the multi-nanowire structure has better measurement sensitivity. However,
under the same sensor cleaning conditions, the reuse characteristics are worse than those
of single nanowire sensors. Resolution of the proposed method can be represented as [26]
S
I
Cres
= In this work, the current
uctuation is approximately 14 nA within 25 s and the results are shown in Figure 11. ccording to Equation (2), the resolution of these sensors can be calculated and are shown
Table 1. Clearly, doped NWGS exhibited higher resolution than undoped NWGS, and
e resolution of NWGS with shorter nanowire length was higher than that of NWGS with
nger nanowire length. An n-type dopant sensor with a nanowire length of 3.5 μm can
hieve the optimum resolution for the proposed sensor which is approximated of 1.75
g/dL. gure 11. Current fluctuation measurement of the proposed system. Figure 11. Current fluctuation measurement of the proposed system. ure 11 Cu e t flu tuatio
ea u e
e t of the
o o ed y te
Figure 11. Current fluctuation measurement of the proposed system. able 1. The resolution of the doped/undoped NWGS. Item
S (μA/(mg/dL))
Theoretical
Practical
I (nA)
Cres (mg/dL)
I (nA)
Cres (mg/dL)
A
0.008
0.1
0.013
14
1.75
B
0.004
0.025
3.5
C
0.002
0.05
7
D
0.001
0.1
14
Table 1. The resolution of the doped/undoped NWGS. Item
S
(µA/(mg/dL))
Theoretical
Practical
∆I (nA)
Cres (mg/dL)
∆I (nA)
Cres (mg/dL)
A
0.008
0.1
0.013
14
1.75
B
0.004
0.025
3.5
C
0.002
0.05
7
D
0.001
0.1
14 ble 1 The resolution of the doped/undoped NWGS
Table 1. The resolution of the doped/undoped NWGS. The threshold voltage (Vth) of nanowire device depends on the doping concentration,
ping types, and the geometric structure of nanowire, such as length, width, and thick-
ss. For nanowire structures with different combinations of length, width, and thickness,
e Vth variation characteristics of the above parameters will also be different. Fong [27]
The threshold voltage (Vth) of nanowire device depends on the doping concentration,
doping types, and the geometric structure of nanowire, such as length, width, and thickness. For nanowire structures with different combinations of length, width, and thickness, the Vth
variation characteristics of the above parameters will also be different. Fong [27] pointed
out that when the length of n-type nanowires was between 1–3 µm and drain voltage
was controlled at 2 V, the change in Vth was proportional to the length of the nanowires. Sensors 2023, 23, 3166 14 of 17 However, when drain voltage was 1 V, the change in Vth has no obvious relationship with
the length of the nanowire. Resolution of the proposed method can be represented as [26]
S
I
Cres
= Figure 12 shows that the single nanowire structure absorbs less
residual gluconic acid, which is significantly better than the multi-nanowire structure. To
prevent the absorption of gluconic acid, the orientation of the microfluidic channel can be
adjusted to align with the orientation of the nanowires. In addition, the injection system
can be replaced by an auto-injector, which provides continuous injection of deionized water
to clean the device. Table 2 summarizes the performance comparison results between the proposed sensor
and the related work cited in Section 1 (in vitro method). The proposed method yields
a reusable glucose sensor with acceptable detection limits, fast response time, and wide
measurement range. It is worth mentioning that the reusable NWGS will help reduce the
manufacturing cost of the sensor, reduce the generation of medical waste, and have less
impact on the environment. Therefore, it will become a green medical product. Table 2. Performance comparison of nanowire glucose sensor. Ref. Detection Limit
Linear Range
Response Time
Reusability
Sample Type
Enzyme
Adopted
[15]
0.16 mM
0–10 mM
X
X
D-glucose
X
[16]
0.1 µM
3 µM–2 mM
X
X
D-glucose
X
[17]
1.31 mM
0–10 mM
>20 s
X
D-glucose/Rabbit
blood serum
X
[18]
1 mM
0–8 mM
X
X
D-glucose
GOx
[19]
3 µM
10 µM–100 mM
X
X
D-glucose
GOx
[20]
1.23 mg/dL
10–300 mg/dL
<5 s
10 times
D-glucose
GOx
This work
1.75 mg/dL
0–400 mg/dL
<5 s
30 times
D-glucose
GOx Table 2. Performance comparison of nanowire glucose sensor. 15 of 17
15 of 17 15 of 17
15 of 17 Sensors 2023, 23, 3166
Sensors 2023, 23, x FOR Figure 12. Top-view SEM images of the proposed sensor after 40 applications. (a) doped type nan-
owire sensors with lengths of 3.5 μm; (b) doped type nanowire sensors with lengths of 5.3 μm. 6 Conclusions
Figure 12. Top-view SEM images of the proposed sensor after 40 applications. (a) doped type
nanowire sensors with lengths of 3.5 µm; (b) doped type nanowire sensors with lengths of 5.3 µm. 6 Conclusions Figure 12. Top-view SEM images of the proposed sensor after 40 applications. (a) doped type nan-
owire sensors with lengths of 3.5 μm; (b) doped type nanowire sensors with lengths of 5.3 μm. 6 Conclusions
Figure 12. Top-view SEM images of the proposed sensor after 40 applications. Resolution of the proposed method can be represented as [26]
S
I
Cres
= (a) doped type
nanowire sensors with lengths of 3.5 µm; (b) doped type nanowire sensors with lengths of 5.3 µm. 6 Conclusions 6. Conclusions
6. Conclusions This work demonstrates the feasibility of a doped/undoped poly-silicon nanowire
sensor for measuring the concentration of glucose. Experimental results show that the res-
olution of the proposed nanowire sensor is strongly related to the length and dopant char-
acteristics of the nanowire. The resolution increases with decreasing nanowire length and
increasing dopant concentration. Moreover, the best resolution is approximately 1.75
mg/dL and the smallest concentration variation that can be determined is 2 mg/dL. This
work further demonstrates the repeatability of the proposed sensor, indicating that the
applications within 30 times can be sustained with an acceptable current-time response
for doped/undoped nanowire sensors. This work demonstrates the feasibility of a doped/undoped poly-silicon nanowire
sensor for measuring the concentration of glucose. Experimental results show that the
resolution of the proposed nanowire sensor is strongly related to the length and dopant
characteristics of the nanowire. The resolution increases with decreasing nanowire length
and increasing dopant concentration. Moreover, the best resolution is approximately
1.75 mg/dL and the smallest concentration variation that can be determined is 2 mg/dL. This work further demonstrates the repeatability of the proposed sensor, indicating that
the applications within 30 times can be sustained with an acceptable current-time response
for doped/undoped nanowire sensors. Author Contributions: Conceptualization, C.-C.H. and C.-L.D.; methodology, C.-C.H. and C.-L.D.;
validation, C.-C.W., W.-K.H. and C.-L.D.; formal analysis, W.-K.H.; investigation, W.-K.H., C.-C.H. and C.-L.D.; resources, C.-C.W.; writing—original draft preparation, C.-C.H.; writing—review and
editing, C.-L.D.; supervision, C.-L.D. and C.-C.H.; project administration, C.-L.D.; funding acquisi-
tion, C.-L.D. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, C.-C.H. and C.-L.D.; methodology, C.-C.H. and C.-L.D.;
validation, C.-C.W., W.-K.H. and C.-L.D.; formal analysis, W.-K.H.; investigation, W.-K.H., C.-C.H. and C.-L.D.; resources, C.-C.W.; writing—original draft preparation, C.-C.H.; writing—review and
editing, C.-L.D.; supervision, C.-L.D. and C.-C.H.; project administration, C.-L.D.; funding acquisition,
C.-L.D. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Science and Technology Council (NSTC) grant
numbers MOST 111-2221-E-005-069 and MOST 110-2634-F-005-006. Funding: This research was funded by National Science and Technology Council (NSTC) grant
numbers MOST 111-2221-E-005-069 and MOST 110-2634-F-005-006. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. 6. Conclusions
6. Conclusions Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Sensors 2023, 23, 3166 16 of 17 Acknowledgments: The authors would like to thank the National Science and Technology Council
(NSTC) of the Republic of China for financially supporting this research under Contract No. MOST
111-2221-E-005-069. This research is also supported (in part) by MOST 110-2634-F-005-006—project
Smart Sustainable New Agriculture Research Center (SMARTer). Conflicts of Interest: The authors declare no conflict of interest. References 1. National Diabetes Statistics Report, 2020 Edition. Available online: https://www.cdc.gov/diabet
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[CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. Department of Electronphysics, National Chiao Tung University, Hsinchu, Taiwan, July 2014.
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Li, J.; Zhang, Y.; To, S.; You, L.; Sun, Y. Effect of nanowire number, diameter, and doping density on nano-FET biosensor sensitivity. References Self-Monitoring Blood Glucose Test Systems for Over-the-Counter Use: Guidance for Industry
and Food and Drug Administration Staff; Food and Drug Administration (FDA): Rockville, MD, USA, 2018. 17 of 17 Sensors 2023, 23, 3166 27. Fong, P.W. A Study on Polycrystalline-Silicon Nanowire Tunnel Thin-Film Transistor with Raised Source/Drain. Master’s Thesis,
Department of Electronphysics, National Chiao Tung University, Hsinchu, Taiwan, July 2014. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
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https://openalex.org/W2553198994
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https://www.frontiersin.org/articles/10.3389/fpls.2016.01705/pdf
|
English
| null |
Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa
|
Frontiers in plant science
| 2,016
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cc-by
| 12,489
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ORIGINAL RESEARCH
published: 18 November 2016
doi: 10.3389/fpls.2016.01705 ORIGINAL RESEARCH
published: 18 November 2016
doi: 10.3389/fpls.2016.01705 Edited by: 1 Queensland Alliance for Agriculture and Food Innovation, University of Queensland, St. Lucia, QLD, Australia, 2 Forest
Industries Research Centre, University of the Sunshine Coast, Maroochydore, QLD, Australia, 3 Forestry & Biosciences,
Agri-Science Queensland, Department of Agriculture and Fisheries, Gympie, QLD, Australia, 4 Prozess Technologie,
St. Louis, MO, USA, 5 Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA, 6 Biological
and Engineering Sciences Center, Sandia National Laboratories, Livermore, CA, USA, 7 Gruppo Ricicla – Biomass and
Bioenergy Laboratory, DiSAA, University of Milan, Milan, Italy Reviewed by:
Liangcai Peng,
Huazhong Agricultural University,
China
Taras P. Pasternak,
Albert Ludwigs University of Freiburg,
Germany In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must
possess a high rate of growth to supply biomass for conversion. Despite the desirability
of a fast growth rate for industrial application, it is unclear what effect growth rate has on
biomass composition or saccharification. We characterized Klason lignin, glucan, and
xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF)
and parental species Corymbia torelliana and C. citriodora subspecies variegata and
measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts
of Klason lignin content (19.7–21.3%) among parental and hybrid populations, whereas
glucan content was clearly distinguished within C. citriodora subspecies variegata (52%)
and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression
indicates that biomass composition is significantly impacted by tree size measured
at the same age, with Klason lignin content increasing with diameter breast height
(DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing
per DBH cm increase (−0.7 and −0.3%, respectively). Polysaccharide content within
C. citriodora subspecies variegata and HF-148 were not significantly affected by tree
size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass
found significant differences among Corymbia populations for total glucose production
from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51
generating the highest amounts of glucose (∼180 mg/g biomass, respectively), with In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must
possess a high rate of growth to supply biomass for conversion. Despite the desirability
of a fast growth rate for industrial application, it is unclear what effect growth rate has on
biomass composition or saccharification. Evaluation of Relationships between
Growth Rate, Tree Size,
Lignocellulose Composition, and
Enzymatic Saccharification in
Interspecific Corymbia Hybrids and
Parental Taxa Adam L. Healey1*, David J. Lee2,3, Jason S. Lupoi4, Gabriella Papa5, Joel M. Guenther5,6,
Luca Corno7, Fabrizio Adani7, Seema Singh5,6, Blake A. Simmons5,6 and
Robert J. Henry1 Adam L. Healey1*, David J. Lee2,3, Jason S. Lupoi4, Gabriella Papa5, Joel M. Guenther5,6,
Luca Corno7, Fabrizio Adani7, Seema Singh5,6, Blake A. Simmons5,6 and
Robert J. Henry1 Edited by: We characterized Klason lignin, glucan, and
xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF)
and parental species Corymbia torelliana and C. citriodora subspecies variegata and
measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts
of Klason lignin content (19.7–21.3%) among parental and hybrid populations, whereas
glucan content was clearly distinguished within C. citriodora subspecies variegata (52%)
and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression
indicates that biomass composition is significantly impacted by tree size measured
at the same age, with Klason lignin content increasing with diameter breast height
(DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing
per DBH cm increase (−0.7 and −0.3%, respectively). Polysaccharide content within
C. citriodora subspecies variegata and HF-148 were not significantly affected by tree
size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass
found significant differences among Corymbia populations for total glucose production
from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51
generating the highest amounts of glucose (∼180 mg/g biomass, respectively), with *Correspondence:
Adam L. Healey
a.healey1@uq.edu.au Specialty section:
This article was submitted to
Plant Biotechnology,
a section of the journal
Frontiers in Plant Science Specialty section:
This article was submitted to
Plant Biotechnology,
a section of the journal
Frontiers in Plant Science
Received: 02 August 2016
Accepted: 31 October 2016
Published: 18 November 2016 Received: 02 August 2016
Accepted: 31 October 2016
Published: 18 November 2016 Edited by:
Chandrashekhar Pralhad Joshi,
Michigan Technological University,
USA INTRODUCTION trial sites indicate C. citriodora subspecies variegata is also
well-suited for pulp and paper production based on predicted
Kraft pulp yield (55% pulp per wood volume) and density
(756 kg/m3), and moderate trait heritability (0.3 and 0.5,
respectively) across multiple trial sites (Brawner et al., 2012). Tree
improvement programs have also demonstrated the potential of
F1 interspecies Corymbia hybrids (C. torelliana × C. citriodora
subspecies variegata), combining desirable forestry traits (form,
wood quality, vegetative propagation) within a single genetic
background and possessing a superior growth rate [127–287%,
diameter breast height (DBH)] as compared to either parental
taxa (Lee et al., 2009). Due to the un-sustainable nature and detrimental effects of
fossil fuels on climate change, there is increased interest for the
development of renewable plant-based alternatives for energy
production (Simmons et al., 2008; Furtado et al., 2014). Advanced
future biofuels will likely derive from non-edible feedstocks,
where structural polysaccharides provide the main substrate
for biochemical conversion into fuel. Lignocellulose, or woody
biomass, is a potential feedstock for bioenergy production
considering its availability, cost of production and scale at which
it can be generated (Wang et al., 2012); however, lignocellulose’s
natural recalcitrance to deconstruction prevents the economic
conversion of biomass into fuel (Studer et al., 2011). While a high rate of growth is desirable for a variety of forestry
applications, it is unclear what effect growth rate plays in altering
biomass composition or affecting enzymatic saccharification. Transgenic manipulation of woody biomass has demonstrated
that alteration of growth rate influences biomass composition
and vice versa. Overexpression of a growth hormone precursor
to gibberellin in transgenic poplar trees significantly improved
growth rate in seedlings and biomass production within stem
tissue. Additionally, transgenic lines also had longer and more
numerous xylem fibers, which are commercially desirable for
producing wood pulp with higher tensile strength (Eriksson
et al., 2000). Disruption of lignin biosynthesis in transgenic
poplar hybrids can negatively impact growth form and habit,
where transgenic lines with significantly reduced lignin content
resulted in brown discolored xylem tissue and dwarfed trees
with reduced height, DBH, and growth rate (Leplé et al.,
2007). Without lignin reinforcement during stem growth and
thickening, xylem fibers are prone to collapse and cavitation
being unable to withstand water pressures required for long
distance transport (Kawaoka et al., 2006; Coleman et al., 2008;
Voelker et al., 2011). INTRODUCTION Similarly, transgenic manipulation of
cellulose biosynthesis can negatively impact growth rate and
alter biomass composition. Joshi et al. (2011) demonstrated that
the up-regulation of cellulose synthase (CesA8) inadvertently
caused sense silencing of the native CesA and transgene,
producing trees with little cellulose (10% dry weight) and
a proportionate increase in lignin content (35%) and non-
cellulosic polysaccharides (55%). As there is a strong interaction
between growth and biomass composition, increased biomass
production could negatively impact biofuel conversion processes
if carbon resources shift toward xylem lignification. Small
percentage increases in lignin content greatly affect substrate
access for cellulases (Gressel, 2008), resulting in biomass that
is inefficiently deconstructed and hydrolyzed. The aim of this
study was therefore to examine the effect of growth rate on
the main components of lignocellulose composition (glucan,
xylan, Klason lignin) and their subsequent effect on enzymatic Enzymatic hydrolysis of structural polysaccharides is the
most critical and costly aspect of biofuel production due to the
structure and chemistry of the plant cell wall (Yu et al., 2011). The major structural components of lignocellulose (cellulose,
hemicellulose, lignin) each contribute to biomass recalcitrance
(Wang et al., 2016), but in woody biomass lignin has been
demonstrated to contribute most negatively to saccharification,
as its structure prevents enzymatic access to cellulose and
non-specifically
binds
and
immobilizes
cellulase
enzymes
(Leu and Zhu, 2013). Additionally, lignin covalently bonds
with hemicellulose, creating lignin-carbohydrate complexes
that
further
inhibit
saccharification
(Yang
and
Wyman,
2004). Hemicellulose also affects efficient saccharification of
lignocellulose due to its composition of difficult to ferment
5′-carbon sugars and its effect on the porosity of biomass
through its cross-linkages with cellulose (Lange, 2007; Nigam
and Singh, 2011). Despite being the main target for conversion
to biofuel, cellulose also resists efficient hydrolysis due to its
crystalline structure resulting in hydrophobic macrofibrils with
limited reactive surface area and extensive hydrogen bonding
(Yang et al., 2011; Mizrachi et al., 2012; Zhang Y. et al., 2015). In order for a lignocellulose feedstock to be considered as
a sustainable option for biofuel production, it must possess a
growth rate suitable for economic harvesting. Eucalypt trees are
an ideal candidate as a biofuel crop based on their established
silviculture practices, global deployment, rapid growth in
marginal soils and wide range of rainfall conditions, and genomic
resources dedicated to wood formation and environmental
resistances (Shepherd et al., 2011; Grattapaglia et al., 2012;
Myburg et al., 2014; Healey et al., 2015). Citation: Healey AL, Lee DJ, Lupoi JS,
Papa G, Guenther JM, Corno L,
Adani F, Singh S, Simmons BA and
Henry RJ (2016) Evaluation
of Relationships between Growth
Rate, Tree Size, Lignocellulose
Composition, and Enzymatic
Saccharification in Interspecific
Corymbia Hybrids and Parental Taxa. Front. Plant Sci. 7:1705. doi: 10.3389/fpls.2016.01705 November 2016 | Volume 7 | Article 1705 1 Frontiers in Plant Science | www.frontiersin.org Healey et al. Impact of Size on Lignocellulose HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based
on
growth
rate,
biomass
composition,
and
further
optimization
of
enzymatic
saccharification yield, high production Corymbia hybrid trees are potentially suitable for
fast-rotation bioenergy or biomaterial production. Keywords: Corymbia, biofuels, eucalypt, saccharification, growth rate, lignin, glucan, xylan INTRODUCTION In Queensland,
Australia, due to climate and environmental stresses, Corymbia
citriodora subspecies variegata (CCV) is the most widely
harvested hardwood tree, based on its form, wood quality and
tolerances to variable soils, drought, pests, and disease (Lee
et al., 2010). Examination of pulpwood traits across multiple November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org 2 Impact of Size on Lignocellulose Healey et al. aid in biomass disruption. Samples were macerated with a glass
rod, covered with aluminum foil, and placed in a 30◦C climate
controlled room for 1 h incubation with mixing every 15 min. After incubation, sulfuric acid was diluted to 4% with ultrapure
water to a final volume of 29 mL. Glass bottles were closed with
a rubber stopper and clamped shut using aluminum crimp top
seals (Sigma-Aldrich). Samples were then autoclaved for 1 h
(121◦C) and allowed to cool before opening. Prior to vacuum
filtration for Klason lignin quantification, 1 mL of decanted
lysate was collected for structural sugars quantification by High
Performance Liquid Chromatography (HPLC). The remaining
supernatant was vacuum filtered through a 25 mL crucible
(Coors #60531), previously heated at 105◦C for a minimum of
1 h and cooled to room temperature for 30 min in a desiccator
before weighing. The serum bottles were washed with deionized
water to remove any particles clinging to the glass wall inner
surface and the solution vacuum filtered free of acid. Crucibles
were dried for a minimum duration of 6 h at 105◦C, and then
cooled to room temperature for 30 min in a desiccator before
crucible weights were collected. saccharification
within
commercial
Corymbia
interspecies
hybrids and parental taxa. Given the significant impact each
biomass component contributes toward saccharification, changes
in biomass composition that occur in response to a high rate
of growth will inform optimal harvest size for industrial use of
woody biomass. Structural Sugars Quantification Structural Sugars Quantification
One hundred and fifty microliters of extracted lysate was filtered
through a 96 well 0.45 µm filter plate (Whatman, 7700-13012)
by centrifugation (3000 × g for 3 min) into a 96 well 200 µL
PCR plate (Bio-Rad, Hercules, CA, USA, HSP9601). PCR plates
were sealed using a pierceable aluminum heat seal (Agilent
06644-001), applied using a PlateLoc sealer (175◦C, 4 s; Agilent
Technologies). HPLC was performed using an Agilent 1260
Infinity system (Agilent, Santa Clara, CA, USA) with a Bio-Rad
87H 300 mm × 7.8 mm Aminex column (Bio-Rad, Hercules,
CA, USA) with a cation H guard column. The refractive index
detector was held at 35◦C. The eluent was 4 mM isocratic sulfuric
acid, prepared with HPLC grade water (Honeywell, Morristown,
NJ, USA) and 98% sulfuric acid (Millipore, Billerica, MA, USA). Each analytical run used an eluent flow rate of 0.6 mL/min, and
temperature set to 60◦C for 16 min. Sugar calibration standards
were prepared and diluted to create an eight-point calibration
curve, 0.015–2.0 mg/mL for cellobiose, xylose, and arabinose,
and 0.03–4.0 mg/mL for glucose. Standards were run at the
beginning, middle and end of each 96 well plate. De-ionized
water blanks were inserted into the sample queue before and
after each run of standards. The concentrations of glucose, xylose,
cellobiose, and arabinose in the samples were calculated using
the Chemstation software package and by integrating the area 2www.gelifesciences.com Sample Collection Populations of Corymbia torelliana, C. citriodora subspecies
variegata and interspecies controlled-cross F1 hybrids (HF) from
the Queensland Department of Agriculture and Fisheries (DAF)
from the Amamoor trial site located near Gympie, Queensland
were measured for DBH at age 13 years. A minimum of five
trees per population per size class (if available) were selected
at random for biomass extraction (Table 1). Wood frass was
collected at a height of 1.3 m on the north-facing side of the tree,
adjusting for knots and tension wood, with a 16 mm wood boring
bit and a modified funnel. Approximately 2–4 g of sapwood
frass was collected per tree, and was air-dried in a paper bag in
an air-conditioned room for 14 days and shipped to the Joint
BioEnergy Institute in Emeryville, CA, USA. Given the large
variation in wood particle size, size reduction was performed
prior to compositional analysis and saccharification by placing
samples into 2 mL polyethylene vials (Sarstedt VWR 72.609.001)
with three ceramic beads (yttrium stabilized zirconia, 5 mm1),
and grinding for 5 min (2.5 min grind, 60s rest, repeat) using
the Joint BioEnergy Institute Biomass Preparation System Robot,
created by Labman Automation Ltd. (North Yorkshire, UK). The
ground biomass was sieved using a 40 mesh (0.4 mm) filter to
further reduce particle size variation. Samples were placed into
re-closable antistatic bags (RoyalBag-#1646) prior to dispensing. The crucibles containing the dried residues were placed in
a furnace and pyrolyzed using a modified pyrolysis protocol
consisting of a 2 h minimum incubation at 575◦C and hold
at 105◦C. The modified protocol was introduced after no
observable or measurable ash content was present after analysing
a representative group of Corymbia biomass samples. Lignin
weight was determined as percent dry biomass as follows: %
Klason lignin = (Final weight after incubation at 105◦C/Initial
weight of samples)∗100. Frontiers in Plant Science | www.frontiersin.org Data Analysis Biomass composition was calculated from the mean of three
technical replicates if the replicate’s coefficient of variation (CV)
was <20%. In instances where CV was greater than 20%, the
mean of two replicates was used. If the CV from all technical
replicates was >20%, all data points were excluded and treated
as missing data. Saccharification values (total glucose production
and conversion efficiency) were calculated from the mean of two
technical replicates if sample CV was <20%. In instances where
CV was >20%, both data points were excluded and treated as
missing data. Multiple linear regression (MLR) was conducted for
each biomass compositional trait and saccharification yield using
R Studio (version 3.0.2), with preliminary analysis to ensure that
there was no violation of the assumption of normality, linearity,
and multicollinearity. Given that XL and XXL trees (Table 1)
could not be found in each Corymbia population, these additional
size classes were initially excluded from the regression models. Compositional Analysis Compositional
Analysis
was
carried
out
using
National
Renewable Energy Laboratory (NREL) methods (Sluiter et al.,
2011) with minor modifications. Using an analytical balance,
100 mg (±5 mg) of biomass was dispensed in triplicate into
100 mL serum bottles. One mL of 72% sulfuric acid was added to
each biomass aliquot, along with a small plastic coated stir-bar to TABLE 1 | Size categories of Corymbia hybrid and parental trees randomly
sampled at age 13 years, from the Amamoor plantation site, located near
Gympie, Australia. Size Class
DBH (cm)
Number of Trees
Small (S)
6.4–12.4
36
Medium (M)
12.5–20.3
39
Large (L)
18.0–24.1
38
Extra Large (XL)
27.0–32.2
23
(excluding Corymbia citriodora
subspecies variegata and HF-151)
Extra Extra Large (XXL)
36.0–40.6
5 (HF-148 only)
Frontiers in Plant Science | www.frontiersin.org TABLE 1 | Size categories of Corymbia hybrid and parental trees randomly
sampled at age 13 years, from the Amamoor plantation site, located near
Gympie, Australia. November 2016 | Volume 7 | Article 1705 3 Healey et al. Impact of Size on Lignocellulose under each sugar peak. Glucan content was calculated as: Glucan
content (%) = ((glucose concentration [mg/mL]∗V∗0.9)/m)∗100
where V is the volume of hydrolysis liquid (mL), m is the mass
of the sample (mg) and 0.9 is the conversion factor for glucose to
glucan. where glucose is glucose concentration in the enzymatic
hydrolysis liquor (mg/mL); V is volume of enzymatic hydrolysis
liquor (mL); m is mass of sample (mg). Biomass Composition Klason lignin, glucan, and xylan content, as well as glucose
yield (mg/g raw biomass) and (%) conversion efficiency are
summarized in Table 2. The population standard deviations
(SDs) for each trait are described below in the text, whereas
experimental standard error (SE) is provided in Table 2. The conversion of cellulose to glucose in the enzymatic
hydrolysis
was
determined
by
the
ratio
of
the
glucose
concentration that was released during enzymatic hydrolysis
to the total glucose in the substrate and was calculated using
formula: Total Glucose Production and Glucan Conversion
γi = µ + Sj + L + εj γi = µ + Sj + L + εj where γ represents enzymatic saccharification [i = total
glucose production (mg glucose/g biomass), theoretical glucan
conversion to glucose (%)], µ represents the intercept of the
model, S represents the various Corymbia populations (j), L is
Klason lignin content (% biomass) of the biomass, and ε is the
vector for random residual error. Enzymatic Saccharification y
Ground biomass samples were dispensed for saccharification
using the Joint BioEnergy Institute Biomass Preparation System
Robot at a target mass of 10 (±0.5) mg of biomass per well
into a 2 mL 96 deep-well polypropylene block (Corning Costar
3961). Samples were dispensed in duplicate between two separate
blocks. Biomass extraction was conducted by adding 1 mL of
80% ethanol into each well using a Biomek FX liquid-handling
robot with an AP96 multichannel pod (Beckman, Coulter, Brea,
CA, USA). Each 96 well block was sealed with a peelable heat
seal and incubated at 37◦C within a thermostatically controlled
room for 24 h with shaking at 150 RPM. Ethanol and extractives
were removed from each well using ultrapure water washes until
the ethanol concentration was less than 1% in a final volume
of 820 µL per well. For hydrothermal pretreatment, plates were
sealed shut with a rubber mat and metal clamp and autoclaved
for 1 h at 121◦C. Sample de-starching prior to hydrothermal
pretreatment and saccharification was found unnecessary as
previous experiments, found no additional sugar release from
Corymbia biomass after amylase treatment (data not shown). Biofuel Trait Model γi = µ + Sj + D + Sj × D + εj where γ represents dependent biomass component (i = Klason
lignin, glucan and xylan (% biomass)), µ represents the intercept
of the model, S represents the various Corymbia populations (j),
D represents measured DBH in cm Sj × D is the interaction term
between species and size and ε is the vector for random residual
error. Saccharification was performed using the Biomek FX to
dispense 180 µL of enzyme solution (8.2:1 v:v ratio of Cellic
CTec2:HTec2; Novozymes, Franklinton, NC, USA) and citrate
buffer (pH 5.0) to a final concentration of 100 mM. Enzyme
loading per well was approximately 60 mg/g glucan, chosen
empirically to maximize the observed differences between
eucalypt samples with known low/high glucose saccharification
yields (data not shown). Each block was sealed with a peelable
seal and incubated at 55◦C for 48 h without agitation in a
Thermos oven (Thermo Scientific, Waltham, MA, USA). After
48 h, the blocks were centrifuged for 3 min at 3000 × g, and
then placed onto the Biomek FX robot which transferred 100 µL
of solution into a Whatman 0.45 µm filter plate. The samples
were centrifuged (3 min at 3000 × g) into a 96 well Bio-Rad
PCR plate and sealed with a pierceable aluminum heat seal and
placed at −80◦C. Prior to running on the HPLC, plates were
thawed overnight at 4◦C and diluted 5X in 100 mM citrate buffer
in a new 96 well PCR plate, sealed with a pierceable aluminum
seal. Glucose quantification using the HPLC was conducted as
previously described for compositional analysis. Total Glucose Production and Glucan Conversion
γi = µ + Sj + L + εj Klason Lignin Content Population
Genetic Background
Lignin
Glucan
Xylan
Glucose Production
Glucose Conversion (%)
Corymbia torelliana (CT)
Mixed Provenances
20.4 (0.4)
35 (2)
13.4 (0.7)
189 (8)
62 (5)
Corymbia citriodora
subspecies variegata (CCV)
W
20.9 (0.6)
52 (2)
20.2 (0.4)
150 (11)
33 (3)
HF-148
CT2-011 × CV2-046
21.0 (0.4)
60.0 (0.9)
20.1 (0.3)
188 (8)
35 (2)
HF-153
CT2-011 × CV2-025
20.7 (0.3)
32 (3)
11 (1)
168 (5)
64 (5)
HF-151
CT2-019 × CV2-018
21.3 (0.4)
38 (4)
13 (1)
169 (6)
56 (6)
HF-51
CT2-002 × CV2-018
19.7 (0.4)
28 (2)
10 (1)
184 (7)
74 (6)
HF-69
CT2-017 × CV2-046
20.7 (0.3)
28 (2)
10.4 (0.7)
149 (8)
63 (5)
Numbers in brackets are the standard errors. W, Woondum Provenance. FIGURE 1 | Klason Lignin content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight
percentage of biomass. Numeric codes for each population are provided in Table 2. Horizontal bars within each boxplot denote the population median with open
circles representing outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. TABLE 2 | Biomass composition (% dry weight basis), glucose yield (mg/g raw biomass) and (%) conversion efficiency obtained from enzymatic
hydrolysis of 13-year-old Corymbia hybrids and parental taxa among small, medium and large size trees. Population
Genetic Background
Lignin
Glucan
Xylan
Glucose Production
Glucose Conversion (%)
Corymbia torelliana (CT)
Mixed Provenances
20.4 (0.4)
35 (2)
13.4 (0.7)
189 (8)
62 (5)
Corymbia citriodora
subspecies variegata (CCV)
W
20.9 (0.6)
52 (2)
20.2 (0.4)
150 (11)
33 (3)
HF-148
CT2-011 × CV2-046
21.0 (0.4)
60.0 (0.9)
20.1 (0.3)
188 (8)
35 (2)
HF-153
CT2-011 × CV2-025
20.7 (0.3)
32 (3)
11 (1)
168 (5)
64 (5)
HF-151
CT2-019 × CV2-018
21.3 (0.4)
38 (4)
13 (1)
169 (6)
56 (6)
HF-51
CT2-002 × CV2-018
19.7 (0.4)
28 (2)
10 (1)
184 (7)
74 (6)
HF-69
CT2-017 × CV2-046
20.7 (0.3)
28 (2)
10.4 (0.7)
149 (8)
63 (5)
Numbers in brackets are the standard errors. W, Woondum Provenance. TABLE 2 | Biomass composition (% dry weight basis), glucose yield (mg/g raw biomass) and (%) conversion efficiency obtained from enzymatic
hydrolysis of 13-year-old Corymbia hybrids and parental taxa among small, medium and large size trees. Klason Lignin Content FIGURE 1 | Klason Lignin content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight
percentage of biomass. Numeric codes for each population are provided in Table 2. Horizontal bars within each boxplot denote the population median with open
circles representing outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. FIGURE 1 | Klason Lignin content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight
percentage of biomass. Numeric codes for each population are provided in Table 2. Horizontal bars within each boxplot denote the population median with ope
circles representing outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. and C. citriodora subspecies variegata (M = 20.9%, SD = 2.5%)
contained greater variation within their Klason lignin content
than each of the hybrid populations HF-148 (M = 21.0%,
SD = 1.5%), HF-151 (M = 21.3%, SD = 1.5%), HF-153
(M = 20.7%, SD = 1.3%), HF-51 (M = 19.7%, SD = 1.2%), and
HF-69 (M = 20.7%, SD = 1.2%) as shown in Figure 1. torelliana Klason lignin content was predicted as equal to 15.6%
+0.32 (DBH). Additionally, within the hybrid population HF-69,
the slope and intercept were significantly different (P < 0.02)
than other Corymbia populations where Klason lignin content
was predicted as equal to 21.0% −0.09 (DBH). Overall, in most
populations Klason lignin content increased by 0.12% per cm
increase of DBH. However, within the Corymbia torelliana
population, Klason lignin content increased by 0.32% per cm
increase of DBH, whereas Klason lignin content decreased by
0.09% per cm increase of DBH within hybrid population HF-69. Multiple
linear
regression
of
Klason
lignin
content
as
predicted
by
DBH
and
population
was
significant
[F(13,92) = 5.02, R2 = 0.34, P = 7.2 × 10−7], with a
significant interaction found between population and DBH
[F(6,92) = 3.77, P = 0.002]. Within populations C. citriodora
subspecies variegata, HF-148, HF-151, HF-153, and HF-51,
Klason lignin content was predicted as equal to 19.0% +0.12
(DBH), where Klason lignin is expressed as percent total biomass
and DBH is measured in cm. Within the Corymbia torelliana
population, the slope and intercept was significantly different
(P < 0.02) to other Corymbia populations, where Corymbia Frontiers in Plant Science | www.frontiersin.org Klason Lignin Content Results from two-step acid hydrolysis show that Corymbia
parental species (Corymbia torelliana and C. citriodora subspecies
variegata) contained similar mean (M) values of Klason lignin
as compared to their F1 interspecies hybrid counterparts. The
parental species Corymbia torelliana (M = 20.4%, SD = 2.0%) Theoretical conversion of glucan to glucose (%)
=
glucose ∗V ∗0.9
glucancontent (%) ∗m
∗
100 Theoretical conversion of glucan to glucose (%)
=
glucose ∗V ∗0.9
glucancontent (%) ∗m
∗
100
Frontiers in Plant Science | www.frontiersin.org Theoretical conversion of glucan to glucose (%) November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org 4 Impact of Size on Lignocellulose Healey et al. TABLE 2 | Biomass composition (% dry weight basis), glucose yield (mg/g raw biomass) and (%) conversion efficiency obtained from enzymatic
hydrolysis of 13-year-old Corymbia hybrids and parental taxa among small, medium and large size trees. Population
Genetic Background
Lignin
Glucan
Xylan
Glucose Production
Glucose Conversion (%)
Corymbia torelliana (CT)
Mixed Provenances
20.4 (0.4)
35 (2)
13.4 (0.7)
189 (8)
62 (5)
Corymbia citriodora
subspecies variegata (CCV)
W
20.9 (0.6)
52 (2)
20.2 (0.4)
150 (11)
33 (3)
HF-148
CT2-011 × CV2-046
21.0 (0.4)
60.0 (0.9)
20.1 (0.3)
188 (8)
35 (2)
HF-153
CT2-011 × CV2-025
20.7 (0.3)
32 (3)
11 (1)
168 (5)
64 (5)
HF-151
CT2-019 × CV2-018
21.3 (0.4)
38 (4)
13 (1)
169 (6)
56 (6)
HF-51
CT2-002 × CV2-018
19.7 (0.4)
28 (2)
10 (1)
184 (7)
74 (6)
HF-69
CT2-017 × CV2-046
20.7 (0.3)
28 (2)
10.4 (0.7)
149 (8)
63 (5)
Numbers in brackets are the standard errors. W, Woondum Provenance. FIGURE 1 | Klason Lignin content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight
percentage of biomass. Numeric codes for each population are provided in Table 2. Horizontal bars within each boxplot denote the population median with open
circles representing outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. TABLE 2 | Biomass composition (% dry weight basis), glucose yield (mg/g raw biomass) and (%) conversion efficiency obtained from enzymatic
hydrolysis of 13-year-old Corymbia hybrids and parental taxa among small, medium and large size trees. Glucan Content Comparison of glucan content within each Corymbia population
revealed
significant
differences
among
samples. Parental
Corymbia torelliana (M = 35%, SD = 10%) and hybrid
populations HF-151 (M = 38%, SD = 13%), HF-153 (M = 32%,
SD = 11%), HF-51 (M = 28%, SD = 7%), and HF-69 (M = 28%, November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org 5 Healey et al. Impact of Size on Lignocellulose FIGURE 2 | Glucan content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight percentage of
biomass. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot denote the population mean with open circles
representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. FIGURE 2 | Glucan content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight percentage of
biomass. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot denote the population mean with open circles
representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. (M = 13%, SD = 4%), HF-51 (M = 10%, SD = 3%), and HF-
69 (M = 10.4%, SD = 2.7%) contained lower amounts of xylan
(Figure 3). Due to the differences in xylan content, populations
C. citriodora subspecies variegata and HF-148 were analyzed
separately from other Corymbia population for the effect of DBH
on xylan content. Analysis of C. citriodora subspecies variegata
and 148 populations found no significant of DBH on xylan
content and no significant differences between populations as
tested with a two-tailed t-test (P > 0.05). SD = 7%) contained similar mean and population variance
for glucan content, while C. citriodora subspecies variegata
(M = 52%, SD = 9%) and population HF-148 (M = 60.0%,
SD = 3.4%) yielded much higher mean amounts of glucan, with
population HF-148 containing the least variation (Figure 2). As
such, these two populations were analyzed separately from other
Corymbia population for the effect of DBH on glucan content. y
p p
g
Analysis of the C. citriodora subspecies variegata population
and hybrid family HF-148 found no significant effect of DBH
on glucan content, but a two-sided t-test found a significant
difference [t(18.7) = 3.46, P = 0.002] in glucan content between
C. Glucan Content citriodora subspecies variegata (M = 52%, SD = 8.2%)
and hybrid family 148 (M = 60.0%, SD = 3.4%). MLR of
the remaining Corymbia populations of glucan content as
predicted by DBH and population was significant [F(5,63) = 4.36,
R2 = 0.20, P = 0.002] with no significant interactions. The
predicted glucan content was equal to 50% −0.7 (DBH), where
glucan content is expressed as percent total biomass and DBH
is measured in cm. Overall, glucan content decreased by 0.7%
for every cm increase of DBH, and the glucan intercept (38%)
for hybrid family HF-69 was significantly lower (P = 0.002) than
other populations. Multiple linear regression of the remaining Corymbia
populations for xylan content as predicted by DBH and
population
was
significant
[F(5,59)
=
5.41,
R2 =
0.26,
P = 0.0004] with no significant interactions. Xylan content
was predicted as equal to 16% −0.3 (DBH), where xylan content
is expressed as percent total biomass and DBH is measured
in cm. In summary, xylan content decreased by 0.3% per cm
increase of DBH. Although DBH significantly effected both
Klason lignin content and structural polysaccharides, separate
linear regressions of Klason lignin content as predicted by glucan
and xylan content were not significant (P > 0.05), suggesting that
polysaccharide content was not significantly affecting Klason
lignin content within Corymbia populations. Xylan Content Multiple linear regression of xylan content within Corymbia
populations (with the exclusion of HF-148) as predicted by
population and DBH was significant [F(7,58) = 6.08, R2 = 0.35,
P = 2.4 × 10−5], with a significant interaction between
population and DBH [F(3,61) = 3.07, P = 0.03]. With the
inclusion of the larger tree populations, xylan content was
predicted as equal to 17% −0.4 (DBH), where xylan content is
expressed as percent biomass and DBH is measured in cm. In
summary, xylan content decreased by 0.4% for each cm increase
of DBH. Within hybrid family 69, xylan content was predicted
as equal to 11% with less effect as DBH increases (−0.1% per cm
increase, P = 0.01). Glucan Conversion Efficiency Comparison of glucose conversion efficiency as expressed as a
percentage of the theoretical conversion of anhydrous glucan
(mg) to glucose (mg), showed significant differences among
Corymbia populations (Figure 6). Population HF-51 (M = 74%,
SD = 17%) underwent the most efficient conversion, followed
by HF-153 (M = 64%, SD = 20%), HF-69 (M = 63%,
SD = 16%), Corymbia torelliana (M = 62%, SD = 21%), HF-
151 (M = 56%, SD = 19%), HF-148 (M = 35%, SD = 4%)
and C. citriodora subspecies variegata (M = 33%, SD = 9%). MLR of glucose conversion as predicted by population and
Klason lignin content was significant [F(6,76) = 7.97, R2 = 0.34,
P = 1.2 × 10−6] with only population differences being
significant. Within Corymbia populations, glucose conversion
was significantly higher (P < 0.01) in populations Corymbia
torelliana, HF-51, HF-151, HF-153, and HF-69 as compared to
C. citriodora subspecies variegata and population HF-148. Klason Lignin Content Multiple linear regression of Klason lignin content as predicted
by population and DBH was significant [F(9,93) = 9.22,
R2 = 0.42, P = 7.1 × 10−10] with a significant interaction
between population and DBH [F(4,93) = 2.50, P = 0.04]. With
the inclusion of the larger trees, Klason lignin content was
predicted as equal to 19.2% +0.11 (DBH), where Klason lignin
content was expressed as percent total biomass and DBH was
measured in cm. Overall, Klason lignin content increased by
0.11% for every cm increase of DBH, whereas hybrid family HF-
69 Klason lignin increased by 0.02% for each cm increase in DBH
(P = 0.02). To investigate which biomass components significantly
effected enzymatic saccharification of hydrothermally pre-
treated
samples,
MLR
of
total
glucose
production
was
completed as predicted by population, Klason lignin content, and
polysaccharide content (either glucan or xylan). Given the strong
(Pearson) correlation between glucan and xylan (r = 0.93), each
term was included separately into the saccharification MLR
model to avoid multicollinearity. Multiple linear regression of total glucose production
as
predicted
by
population,
Klason
lignin
content
and
polysaccharide
content
(glucan
or
xylan)
was
significant
[F(7,82) = 15.82, R2 = 0.54, P = 5.7 × 10−13], however,
as glucan content, xylan content and interactions between
explanatory variables were not significant (P > 0.05), these terms
were removed from the final model. Total glucose production
was predicted as equal to 410.5 −10.6 (Klason lignin), where
glucose production was measured as mg of glucose released
per g of pretreated biomass and Klason lignin was measured
as percent total biomass. In summary, glucose production
decreased by 10.6 mg for each percentage increase of Klason
lignin content. The regression intercepts for populations HF-153
(387 mg/g), C. citriodora subspecies variegata (354 mg/g) and
HF-69 (366 mg/g) were significantly lower (P < 0.02) than other
Corymbia populations (Figure 5). Glucan Content Multiple linear regression of the Corymbia populations for
glucan content (again excluding population HF-148) as predicted
by population and DBH was also significant [F(7,65) = 3.44,
R2 = 0.19, P = 0.003], with a significant interaction found
between population and DBH [F(3,65) = 3.92, P = 0.01]. With
the inclusion of the larger trees, glucan content was predicted
as equal to 48% −0.9 (DBH), where glucan content is expressed
as percent biomass and DBH is measured in cm. Overall, glucan
content decreased by 0.9% for each cm increase of DBH, whereas
hybrid family HF-69 glucan content increased by 0.3% per cm
increase of DBH (P = 0.001). DISCUSSION In this study, determination of biomass composition of Corymbia
F1 interspecies hybrids and parental species was completed using
the NREL Laboratory Analytical Procedure-Determination of
Structural Carbohydrates and Lignin in Biomass (Sluiter et al.,
2011), which corrects for ash content within acid insoluble
residue measured gravimetrically after pyrolysis at 575◦C. During
testing and optimization of this procedure, ash content within
Corymbia samples was below an amount that could be reliably
measured with an analytical balance, resulting in a protocol
modification that shortened the pyrolysis step to clean crucibles
before the next use. Ash content is detrimental to liquid fuel
conversion processes (as non-biodegradable residue) as well Enzymatic Saccharification
Total Glucose Production Analysis of xylan content found the same trend as glucan content,
with parental C. citriodora subspecies variegata (M = 20.2%,
SD = 1.7%) and hybrid family HF-148 (M = 20.1, SD = 1.1%)
containing highest mean xylan amounts. By comparison,
the
remaining
Corymbia
populations
Corymbia
torelliana
(M = 13.4%, SD = 3.3%), HF-153 (M = 11%, SD = 4%), HF-151 Analysis of total glucose production (mg glucose/g biomass) after
enzymatic saccharification of hydrothermally pretreated biomass
found significant differences in glucose release from populations
of Corymbia. Comparison of population means found that November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org 6 Impact of Size on Lignocellulose Healey et al. to see whether the same trends continued at larger DBH
sizes. parental Corymbia torelliana (M = 189, SD = 39) and hybrid
populations HF-148 (M = 188, SD = 24) and HF-51 (M = 184,
SD = 24) released the highest amounts of glucose from biomass,
followed by hybrid populations HF-151 (M = 169, SD = 25),
HF-153 (M = 168, SD = 21), C. citriodora subspecies variegata
(M = 150, SD = 36) and HF-69 (M = 149, SD = 25) (Figure 4). Additional Size Category Analysis Although populations of C. citriodora subspecies variegata and
hybrid family HF-151 did not contain trees with DBH beyond the
large size class, the remaining Corymbia populations contained
trees of much larger DBH. Given the significant effect of size
on biomass composition, populations including larger trees (XL)
and in the case of population HF-148, (XXL trees) (Table 1)
were re-analyzed for Klason lignin, glucan and xylan content November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org 7 Healey et al. Impact of Size on Lignocellulose FIGURE 3 | Xylan content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight percentage of
biomass. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot denote the population mean with open circles
representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. FIGURE 3 | Xylan content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight percentage of
biomass. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot denote the population mean with open circles
representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. FIGURE 4 | Glucose released from enzymatic saccharification of Corymbia hybrids and parental taxa at age 13 years, expressed in mg of glucose per
g of biomass. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot denote the population mean with open circles
representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. as negatively affecting the calorific value of wood and plant
If an advanced biofuel feedstock to be considered sustainable FIGURE 3 | Xylan content of interspecies Corymbia hybrid populations and parental taxa at age 13 years, expressed as dry weight percentage of
biomass. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot denote the population mean with open circles
representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. Additional Size Category Analysis Glucose production is expressed as mg of glucose released
after 48 h of enzymatic hydrolysis per g of raw biomass and Klason lignin is expressed as a percentage of dry biomass. Numeric codes for each population are
included within Table 2. Each colored regression line represents populations whose intercept is significantly different (P < 0.05) from the overall regression (black
line). CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. FIGURE 6 | Glucan conversion efficiency from enzymatic saccharification of Corymbia hybrids and parental taxa at age 13 years, expressed as a
percentage of mg of glucose released per mg of glucan. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot
denote the population mean with open circles representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata;
CT, Corymbia torelliana. During the growth and expansion of the plant cell wall,
shifting carbon resources can simultaneously increase lignin
content while decreasing polysaccharide content. This has been
in increased cellulose content, biomass and growth rate (Hu
et al., 1999), disruption of lignin biosynthesis typically results
in negative pleiotropic effects on growth and form. For FIGURE 5 | Total glucose production as predicted by population and Klason lignin content. Glucose production is expressed as mg of glucose released
after 48 h of enzymatic hydrolysis per g of raw biomass and Klason lignin is expressed as a percentage of dry biomass. Numeric codes for each population are
included within Table 2. Each colored regression line represents populations whose intercept is significantly different (P < 0.05) from the overall regression (black
line). CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. FIGURE 5 | Total glucose production as predicted by population and Klason lignin content. Glucose production is expressed as mg of glucose released
after 48 h of enzymatic hydrolysis per g of raw biomass and Klason lignin is expressed as a percentage of dry biomass. Numeric codes for each population are
included within Table 2. Each colored regression line represents populations whose intercept is significantly different (P < 0.05) from the overall regression (black
line). CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. FIGURE 6 | Glucan conversion efficiency from enzymatic saccharification of Corymbia hybrids and parental taxa at age 13 years, expressed as a
percentage of mg of glucose released per mg of glucan. Numeric codes for each population are included within Table 2. Additional Size Category Analysis ep ese
g suspec ed ou e s
5
ou s de
e
e qua
e a ge CC , Co y
b a c
odo a subspec es a ega a; C , Co y
b a o e a a
FIGURE 4 | Glucose released from enzymatic saccharification of Corymbia hybrids and parental taxa at age 13 years, expressed in mg of glucose per
g of biomass. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot denote the population mean with open circles
representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. FIGURE 4 | Glucose released from enzymatic saccharification of Corymbia hybrids and parental taxa at age 13 years, expressed in mg of glucose per
g of biomass. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot denote the population mean with open circles
representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata; CT, Corymbia torelliana. as negatively affecting the calorific value of wood and plant
processing costs from thermo-chemical conversion (McKendry,
2002; Jørgensen et al., 2007). Eucalyptus and Corymbia have been
characterized as possessing low ash content (<1%), (Guerrero
et al., 2005; Magalhães et al., 2011; Çetrinköl et al., 2012), which
decreases with tree age (Kumar et al., 2010). as negatively affecting the calorific value of wood and plant
processing costs from thermo-chemical conversion (McKendry,
2002; Jørgensen et al., 2007). Eucalyptus and Corymbia have been
characterized as possessing low ash content (<1%), (Guerrero
et al., 2005; Magalhães et al., 2011; Çetrinköl et al., 2012), which
decreases with tree age (Kumar et al., 2010). If an advanced biofuel feedstock to be considered sustainable,
it must possess a growth rate that warrants continued economic
harvesting of that crop (Hinchee et al., 2009). Despite
the advantages of lignocellulose for biofuel production, the
presence of lignin and the biomass’ natural recalcitrance are
substantial barriers to overcome, before this can be realized. November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org Frontiers in Plant Science | www.frontiersin.org 8 Healey et al. Impact of Size on Lignocellulose FIGURE 5 | Total glucose production as predicted by population and Klason lignin content. Additional Size Category Analysis This has resulted in
development of a variety of pretreatments for eucalypt biomass,
to increase efficiency of enzymatic hydrolysis (Yu et al., 2010;
Silva et al., 2011; Papa et al., 2012; Santos et al., 2012; Yáñez-S
et al., 2013; Zhang C. et al., 2015). Lignin removal also creates
pores in the cell wall through which cellulases can gain access to
cellulose microfibrils (Yu et al., 2011). The pretreatment method
for this dataset was hydrothermal (pressurized hot-water),
which does not remove lignin but solubilizes hemicellulose,
and disrupts the cellulose-hemicellulose-lignin complex. In this
study, nor glucan or xylan content significantly impacted total
glucose production from biomass. Cellulose, the primary donor
of glucose during saccharification, resists enzymatic hydrolysis
through hydrogen bonding and microfibril crystallinity. The
highly compact cellulose polymer is hydrophobic, so only
the hydrophilic ends of the microfibril are susceptible to
enzymatic attack. Without disruption of the microfibril structure
(normally achieved through energy intensive ball-milling) which
increases porosity and promotes cellulose accessibility, glucan
content independently considered may not significantly affect
saccharification (Leu and Zhu, 2013) or ethanol production
during simultaneous saccharification and fermentation (Vinzant Given the economic importance of eucalypt taxa for industrial
processes such as pulp and paper, their biomass composition
has been well researched (Table 3). Klason lignin content
among the Corymbia populations is consistent with values
found in the literature and while acid soluble lignin was
not measured here, it is reasonable to expect a similar
range (2–4%) that would also likely have a detrimental effect
on saccharification and subsequent fermentation (Ximenes
et al., 2010). While the xylan content among Corymbia
populations is consistent with those found in the literature
for eucalypts, mean glucan content within Corymbia torelliana
and hybrid families HF-153, HF-51, and HF-69 are low in
comparison to HF-151, C. citriodora subspecies variegata and
HF-148. While mean glucan content in C. citriodora subspecies
variegata and HF-148 samples is higher than other Corymbia
populations or other literature values, the result is consistent with
α-cellulose content (cellulose which remains insoluble) within
elite Eucalyptus hybrids (E. urophylla × E. grandis) (Shinya et al.,
2016). In their study Shinya et al. (2016) evaluated the wood
properties of 918 hybrids and selected two genotypes (AM380
and AM063) for their extreme Klason lignin content (35 and
20%, respectively). Further characterization of AM063 biomass
estimated its α-cellulose content was 59% dry weight (native
wood). Additional Size Category Analysis Alternatively,
overexpression of gibberellin 20-oxidase (a precursor to the
gibberellin hormone) in hybrid poplars resulted in trees with
faster growth in height and DBH, increased biomass, and
more numerous and longer xylem fibers (Eriksson et al., 2000). Considering the increased biomass production of the Corymbia
hybrid populations (Lee et al., 2009), it is reasonable to expect
increased lignification of xylem tissue correlates with growth,
consistent with wood samples taken across each size category
(Table 1). et al., 1997). Additionally, an increase in hemicellulose content
has been demonstrated to disrupt cellulose crystallinity in
Miscanthus,
thereby
increasing
enzymatic
hydrolysis
after
acidic and alkaline pretreatment (Xu et al., 2012), the opposite
effect has been demonstrated in Poplar transgenic experiments,
where
disruption
of
glycosyltransferase
GAUT12
resulted
in transgenic trees with less xylan (17–30% reduction) and
increased saccharification yield (4–8% increase in glucose
recovery) without a significant reduction in lignin content
(Biswal et al., 2015). Future studies of this nature would benefit
from the investigation of the transcriptome within the natural
trait extremes within populations that cannot yet be transformed
in order to discern the genetic mechanisms by which trees
compensate for an increased growth rate. p
g
Within Corymbia samples, the highest conversion efficiency
was achieved within population HF-51 (M = 74%), with
populations
HF-153,
HF-51,
and
Corymbia
torelliana
all
containing samples that approached 100% conversion (Figure 6). While higher glucan conversion has been achieved in the
literature (Sykes et al., 2015), our intent was to maximize
the relative differences among parental species and F1 hybrid
populations. C. citriodora subspecies variegata and HF-148
underwent the least efficient conversion of glucan to glucose,
unsurprising considering the fixed enzyme dosage, however, HF-
148 released the highest mean glucose amount from biomass,
a promising result for future investigation with alternative
pretreatments designed to increase cellulose accessibility and
saccharification. Enzymatic
saccharification
of
Corymbia
biomass
demonstrates the strong negative effect lignin content on
enzymatic hydrolysis. This is attributed to the structure of lignin
physically inhibiting enzymatic access to cellulose microfibrils,
forming cross-linkages with hemicellulose, and lignin non-
specifically binding and immobilizing cellulases (Yu et al., 2011;
Zhao et al., 2012; Leu and Zhu, 2013). Without delignification,
up to 70% of cellulases remain immobilized within lignin
(Berlin et al., 2005; Jørgensen et al., 2007). Additional Size Category Analysis Horizontal bars within each boxplot
denote the population mean with open circles representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata;
CT, Corymbia torelliana. FIGURE 6 | Glucan conversion efficiency from enzymatic saccharification of Corymbia hybrids and parental taxa at age 13 years, expressed as a
percentage of mg of glucose released per mg of glucan. Numeric codes for each population are included within Table 2. Horizontal bars within each boxplot
denote the population mean with open circles representing suspected outliers 1.5X outside the interquartile range. CCV, Corymbia citriodora subspecies variegata;
CT, Corymbia torelliana. During the growth and expansion of the plant cell wall,
shifting carbon resources can simultaneously increase lignin
content while decreasing polysaccharide content. This has been
demonstrated through transgenic manipulation of lignocellulose
biosynthesis. While in some instances, lignin reduction results During the growth and expansion of the plant cell wall,
shifting carbon resources can simultaneously increase lignin
content while decreasing polysaccharide content. This has been
demonstrated through transgenic manipulation of lignocellulose
biosynthesis. While in some instances, lignin reduction results in increased cellulose content, biomass and growth rate (Hu
et al., 1999), disruption of lignin biosynthesis typically results
in negative pleiotropic effects on growth and form. For
example, suppression of the LIM domain transcription factor
in Eucalyptus camaldulensis, an positive regulator of several November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org 9 Impact of Size on Lignocellulose Healey et al. lignin biosynthesis genes, resulted in a transgenic lines with
reduced lignin content (17% as compared to 24%-wild-type
[WT]) that frequently dropped upper leaves (Kawaoka et al.,
2006). Specific targeting of the phenylpropanoid cinnamoyl-CoA
reductase (CCR) gene in transgenic poplars, produced trees with
less lignin (17% vs. 21%-WT) that that had significantly reduced
growth (height, DBH, and growth rate) (Leplé et al., 2007). Additionally, disruption of lignin biosynthetic pathways often
accompanies increased deposition of phenolics and extractives
within wood tissue resulting in discolouration. Down-regulation
of 4-coumarate:coenzymeA ligase (4CL) in poplar also results in
reduced total lignin content and discolouration of xylem tissues,
with biomass and leaf area reduced by half as compared to WT
(Voelker et al., 2011). Adequate lignification of xylem vessels
allows long distance transport of water through maintenance
of internal water tension. Irregular xylem formation causes
vasculature collapse, inadequate water transport, and weakened
carbon sink strength (Coleman et al., 2008). Frontiers in Plant Science | www.frontiersin.org Additional Size Category Analysis Similarly, the α-cellulose content of genotype AM380
was estimated at 48%, suggesting a negative correlation between
lignin and cellulose. While the results glucan content from
C. citriodora subspecies variegata and HF-148 were consistent
with Shinya et al. (2016), the negative (Pearson) correlation November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org 10 Impact of Size on Lignocellulose Healey et al. content in Corymbia hybrids HF-153, HF-51, HF-69 resembled
the Corymbia torelliana parental species, whereas HF-151 was
typically intermediate, and HF-148 was transgressive for glucan
content (Figure 2). Other investigation of stem and leaf attributes
among controlled-cross F1 Corymbia torelliana × section
Maculatae crosses (to which C. citriodora subspecies variegata
belongs), hybrids typically resembled the Corymbia torelliana
maternal parent or were transgressive (Abasolo et al., 2012). Based on maternal parentage and the moderate to high
heritability of cellulose content (h2 = 0.42–0.86) (Kube et al.,
2001; Apiolaza et al., 2005; Stackpole et al., 2010) it would be
expected that Corymbia hybrids with shared genetics (HF-148
and HF-153) should be similar in structural polysaccharide
content. This, however, is not the case. Additionally, for the between Klason lignin and glucan content within this dataset
(r = −0.1) was not significant (P > 0.05). Despite cellulose being
the principle contributor of glucan released from lignocellulose,
the primary cell wall of woody species also contains xyloglucan, a
matrix polysaccharide which contains glucan and xylan residues
(Carpita and Gibeaut, 1993; Harris and DeBolt, 2010). If the high
% dry weight of xylan within C. citriodora subspecies variegata
and HF-148 can be attributed to xyloglucan, then it would be
reasonable to expect that glucan content could also be increased. between Klason lignin and glucan content within this dataset
(r = −0.1) was not significant (P > 0.05). Despite cellulose being
the principle contributor of glucan released from lignocellulose,
the primary cell wall of woody species also contains xyloglucan, a
matrix polysaccharide which contains glucan and xylan residues
(Carpita and Gibeaut, 1993; Harris and DeBolt, 2010). If the high
% dry weight of xylan within C. citriodora subspecies variegata
and HF-148 can be attributed to xyloglucan, then it would be
reasonable to expect that glucan content could also be increased. F1 generation hybrids are typically intermediate with regard
to parental trait values, but occasionally hybrids will either
resemble one parent or exceed both (Rosenthal et al., 2002;
Barbour et al., 2003). Additional Size Category Analysis Despite lignin content of hybrid populations
being intermediate to parental species, structural polysaccharide TABLE 3 | Composition of major structural components of eucalypt biomass. Composition (%)
Species
Glucan
Xylan
Klason Lignin
Acid Soluble Lignin
Reference
E. globulus
46.1
14
20.9
3.0
Santos et al., 2011
E. nitens
41.8
15.9
22.3
3.2
E. urophylla × E. grandis
48.5
10.7
24.5
2.1
E. grandis
44.9
11.4
26.2
–
Yu et al., 2010
E. globulus
46.3
16.6
23
3.5
Garrote et al., 2007
E. grandis
44.6
15.33
25.8
–
Emmel et al., 2003
E. dunnii
47.5
17.31
27
3.4
McIntosh et al., 2012
C. citriodora subspecies variegata
48.5
17.1
24.36
4.19
E. urophylla × E. grandis
59
–
20
–
Shinya et al., 2016
FIGURE 7 | Historical Corymbia growth measurements taken from the Amamoor plantation. Measurements of diameter breast height (DBH) were taken at
years 1.9, 2.9, 5.6, and 8.6 from planting. Frontiers in Plant Science | www.frontiersin.org
11
November 2016 | Volume 7 | Article 1705 TABLE 3 | Composition of major structural components of eucalypt biomass. Composition (%)
Species
Glucan
Xylan
Klason Lignin
Acid Soluble Lignin
Reference
E. globulus
46.1
14
20.9
3.0
Santos et al., 2011
E. nitens
41.8
15.9
22.3
3.2
E. urophylla × E. grandis
48.5
10.7
24.5
2.1
E. grandis
44.9
11.4
26.2
–
Yu et al., 2010
E. globulus
46.3
16.6
23
3.5
Garrote et al., 2007
E. grandis
44.6
15.33
25.8
–
Emmel et al., 2003
E. dunnii
47.5
17.31
27
3.4
McIntosh et al., 2012
C. citriodora subspecies variegata
48.5
17.1
24.36
4.19
E. urophylla × E. grandis
59
–
20
–
Shinya et al., 2016
FIGURE 7 | Historical Corymbia growth measurements taken from the Amamoor plantation. Measurements of diameter breast height (DBH) were taken at
years 1.9, 2.9, 5.6, and 8.6 from planting. Frontiers in Plant Science | www.frontiersin.org
11
November 2016 | Volume 7 | Article 1705 LE 3 | Composition of major structural components of eucalypt biomass. TABLE 3 | Composition of major structural components of eucalypt biomass. g a d s
6
5 33
5 8
e et a ,
003
E. dunnii
47.5
17.31
27
3.4
McIntosh et al., 2012
C. citriodora subspecies variegata
48.5
17.1
24.36
4.19
E. urophylla × E. grandis
59
–
20
–
Shinya et al., 2016
FIGURE 7 | Historical Corymbia growth measurements taken from the Amamoor plantation. Additional Size Category Analysis Measurements of diameter breast height (DBH) were taken at
years 1.9, 2.9, 5.6, and 8.6 from planting. FIGURE 7 | Historical Corymbia growth measurements taken from the Amamoor plantation. Measurements of diameter breast height (DBH) were taken at
years 1.9, 2.9, 5.6, and 8.6 from planting. November 2016 | Volume 7 | Article 1705 11 Frontiers in Plant Science | www.frontiersin.org Impact of Size on Lignocellulose Healey et al. FUNDING This work was part of the DOE Joint BioEnergy Institute
(http://www.jbei.org) supported by the U.S. Department of
Energy, Office of Science, Office of Biological and Environmental
Research, through contract DE-AC02-05CH11231 between
Lawrence Berkeley National Laboratory and the U.S. Department
of Energy. ACKNOWLEDGMENTS The authors would like to thank David Osvorne of USC and
Danica Pratt for assisting with wood collection and DBH
measurements and wood collection at the Amamoor plantation
site. Additionally, thank you to Guilherme Batista, Suellen
da Silva and Evelin Verdolin Brandao for their hard work
and assistance with compositional analysis and saccharification. The United States Government retains and the publisher, by
accepting the article for publication, acknowledges that the
United States Government retains a non-exclusive, paid-up,
irrevocable, worldwide license to publish or reproduce the
published form of this manuscript, or allow others to do so, for
United States Government purposes. CONCLUSION As advanced future biofuel feedstocks require a high rate
of growth to justify harvesting at an industrial scale and
efficient deconstruction and conversion is dependent on biomass
composition, a key consideration for a renewable feedstock
is the impact of growth on lignocellulose formation. This
investigation identified that in response to growth in Corymbia
populations, major structural components of biomass were
significantly impacted by tree size, shifting toward increased
recalcitrance through increased Klason lignin content and
deceased polysaccharide content. This research suggests that
fast growing trees harvested under fast rotations would be
best suited for lignocellulosic biofuel production. Given current
forestry management practices involve thinning trees planted
at a high stocking rate to promote growth in high value
trees, traditional forestry and bioenergy applications could be
combined if thinned trees are removed for biofuel use before
lignification is complete. AUTHOR CONTRIBUTIONS hybrid families that have at least one parent in common
(HF-148
and
HF-69;
HF-51
and
HF-151)
parentage
of
the common male pollen parent among the hybrids failed
to
elucidate
genetic
patterns
underlying
polysaccharide
content. Population HF-148 is clearly distinguished from
the other hybrids by having transgressive glucan content,
while historical investigation of data collected from the
Amamoor plantation site also confirms heterosis for growth
rate (Figure 7). Between ages 1.9 and 8.6, each hybrid family
at Amamoor, in terms of DBH, outperformed parental species
with HF148 distinguished having the fastest growth beyond
age 2.9. AH: Responsible for overall experimental design, sample
collection, analysis and manuscript writing. DL: Responsible for
overall experimental design, sample collection, interpretation
of
results
and
manuscript
editing. JL:
Responsible
for
overall experimental design, processing samples for analysis,
interpretation
of
results
and
manuscript
writing. GP:
Responsible for two-step acid hydrolysis design, collecting
and interpreting composition data, and manuscript writing. JG: Responsible for high-throughput saccharification design,
collecting and interpreting glucose data, and manuscript editing. LC: Responsible for high-throughput design and collection
of structural polysaccharide data and manuscript editing. FA: Responsible for hydrolysis and saccharification design,
interpretation of results and manuscript editing. SS: Responsible
for two-step acid hydrolysis design, interpretation of results
and manuscript editing. BS: Responsible for hydrolysis and
saccharification design, interpretation of results and manuscript
editing. RH: Responsible for overall experimental design,
interpretation of results and manuscript writing. All authors have
agreed on the final version of this manuscript and are accountable
for the research therein. g
Specific consideration of hybrid family HF-69 found each of
its traits relating to biomass composition responded significantly
different from other Corymbia populations. With the inclusion
of the XL DBH tree data, Klason lignin, glucan, and xylan
content as predicted by DBH of HF69 was less impacted
by increasing tree size. Interestingly, evaluation of historical
growth records from the Amamoor plantation also indicate HF-
69 displayed an odd growth habit, displaying nearly double
the variation in DBH of any other Corymbia population at
age 8.6 (Figure 7). While HF-69 was not a top performer
regarding total glucose production (M = 149 mg/g biomass)
or glucan to glucose conversion (M = 63%), its biomass
stability and large growth variation for selection and improved
breeding may be desirable for other forest industries, such
as timber production, where product consistency is highly
regarded. Barbour, R. C., Potts, B. M., and Vaillancourt, R. E. (2003). Gene flow between
introduced and native Eucalyptus species: exotic hybrids are establishing in the
wild. Aust. J. Bot. 51, 429–439. doi: 10.1071/BT03016
Berlin, A., Gilkes, N., Kurabi, A., Bura, R., Tu, M., Killburn, D., et al. (2005). Weak
Lignin-binding enzymes. A Novel Approach to Improve Activity of Cellulose
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10.1016/j.enzmictec.2009.11.001 Copyright © 2016 Healey, Lee, Lupoi, Papa, Guenther, Corno, Adani, Singh,
Simmons and Henry. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms. Xu, N., Zhang, W., Ren, S., Liu, F., Zhao, C., Liao, H., et al. (2012). Hemicelluloses
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ethanol production from Eucalyptus globulus wood by autocatalized organosolv November 2016 | Volume 7 | Article 1705 Frontiers in Plant Science | www.frontiersin.org 14
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Disentangling institutions: a challenge
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To cite this version: Claude Ménard. Disentangling institutions: a challenge. Agricultural and Food Economics, 2022, 10
(1), pp.16. 10.1186/s40100-022-00223-w. hal-04012202 Disentangling institutions: a challenge Claude Ménard* That “institutions matter” has become a mantra among economists. It has not always
been so. For a long time, the conventional wisdom considered institutions as exogenous
parameters, the study of which should be delegated to ‘soft’ social sciences, mainly soci-
ology and political sciences. And many contemporary economists still disregard the
analysis of institutions in their research agenda, mainly because of the difficulty in quan-
tifying and modeling their role. Centre d’Economie de La
Sorbonne, University of Paris,
Paris, France However, this state of affair is changing. The breakthrough came from the pioneer-
ing contributions of Ronald Coase, who showed that transaction costs permeate all
economic activities and that these costs largely depend on institutional factors, e.g., the
definition and implementation of property rights and more generally the legal regime. Three followers, also Nobel winners, pushed the analysis further. Douglass North sub-
stantiated the role of macro-institutions, namely: the polity and the judiciary, in under-
standing the drivers of growth and development as well as the potential obstacles
institutions may create, keeping nations poor. Oliver Williamson rejuvenated organiza-
tion theory, showing the determining role of transaction costs in the choice of micro-
institutional arrangements and extending the set of solutions beyond markets and
hierarchies through the introduction of hybrid solutions and the role of contracts. Last,
Elinor Ostrom deepened our understanding of issues of governance when it comes to
public goods and the commons, pointing out the key role of collective action and the
complex conditions of its success. These contributors and their followers initiated the
development of an integrated set of concepts and local models (for an extensive review,
see Ménard and Shirley 2022). Notwithstanding these analytical progresses, going far beyond the vague statement
that ‘institutions matters’, a major gap subsists when it comes to connecting macro-
institutions (often assimilated to the ‘northian’ branch) and micro-institutions (the ‘wil-
liamsonian’ branch). If we define macro-institutions (the “institutional environment” in
Davis and North, 1971: 6) as the layer within which the rules of the game and the general
conditions of their implementation are established, and micro-institutions (“the “insti-
tutional arrangements,” ibid.) as the layer within which transactions are organized and
the allocation and usage of resources shaped, what are the transmission mechanisms
linking these two dimensions? And what role for norms and values, which North tagged
as “informal rules”? © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits
use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third
party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate-
rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or
exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
creativecommons.org/licenses/by/4.0/. Open Access Open Access HAL Id: hal-04012202
https://hal.science/hal-04012202v1
Submitted on 2 Mar 2023 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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publics ou privés. HAL is a multi-disciplinary open access
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entific research documents, whether they are pub-
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution - NonCommercial - NoDerivatives 4.0
International License Distributed under a Creative Commons Attribution - NonCommercial - NoDerivatives 4.0
International License Ménard Agricultural and Food Economics (2022) 10:16
https://doi.org/10.1186/s40100-022-00223-w Agricultural and Food
Economics Disentangling institutions: a challenge Very little is known about these modalities of interaction besides
some notable exceptions in the literature on regulation. What is missing is an integrative Ménard Agricultural and Food Economics (2022) 10:16 Page 2 of 3 model to capture the modalities of interdependence/interactions between macro- and
micro-institutions. This lacuna might also contribute to the poor state of affair when it
comes to measuring the impact of institutions on economic performance.1 Think about the issue of sustainability, a polysemic word that makes the phenomenon
it intends to capture hard to grasp. Researchers in the field have primarily focused their
attention on the macro-layer of public policies and/or the micro-layer of actors devel-
oping innovative strategies to “support sustainable, resilient, and food-secure systems”
(Grando and Brunori 2020: 5). However, very little is said about the devices and mecha-
nisms through which public policies and the strategies of actors interact. For example,
how is a regulation intending to guarantee food safety implemented among actors of a
supply chain spread over several countries with different political and judicial regime? f
Recent contributions intend to fulfill this challenging gap. Although built on different
preliminaries, they concur in emphasizing the key role played by “intermediate” (e.g.,
Abbott et al. 2017), “meso-institutions” (e.g., Kunneke et al. 2021) in the implementa-
tion and adaptation of rules and norms that connect the macro- and micro-institutional
layers. Meso-institutions bridge the gap through the accomplishment of four main func-
tions. (1) Meso-institutions translate and adjust general rules to specific spatial–tempo-
ral conditions. Consider EU regulation intending to support sustainability by restricting
the usage of certain pesticides. These general rules need adaptation to specific crops,
climatic conditions, etc. In the EU this is processed through dedicated national agencies
(e.g., ANSES in France, BfR in Germany, ISS in Italy). In that process, even translation
of rules in different languages matter. (2) Meso-institutions monitor the actual imple-
mentation of rules. For example, laws or directives might specify that environmental
goals should be reached through contracts, while the actual design and implementation
of these contracts is delegated to parties to the supply chain. In the Netherlands, EU’s
agri-environmental schemes are implemented through the intermediation of collective
of farmers contracting with the Dutch RVO and NVWA agencies. (3) Meso-institutions
also enforce rules, typically through penalties and rewards that motivate or even con-
strain micro-institutional actors to conform. 1 As one referee pointed out, it might also partially explain why the study of micro-institutions assumes most of the time
a given macro-institutional environment, and why comparative studies of micro-institutions across different environ-
ments are so scarce. References References
Abbott KW, Levi-Faur D, Sindal D (2017) Theorizing regulatory intermediaries: the RIT model. Ann Am Acad Polit Soc Sci
670:14–35
Davis L, North DC (1971) Institutional change and American economic growth. Cambridge University Press, Cambridge
(UK)
Grando S, Brunori G (eds) (2020) Innovation for sustainability. Research in rural sociology and development series, vol 25. Emerald Publishing Limited, Bradford
Kunneke R, Ménard C, Groenewegen J (2021) Network Infrastructures: technology meets Institutions. Cambridge Univer-
sity Press, Cambridge
Ménard C, Shirley M (2022) Advanced introduction to new institutional economics. Edward Elgar Publishing, Cheltenham References
Abbott KW, Levi-Faur D, Sindal D (2017) Theorizing regulatory intermediaries: the RIT model. Ann Am Acad Polit Soc Sci
670:14–35
Davis L, North DC (1971) Institutional change and American economic growth. Cambridge University Press, Cambridge
(UK)
Grando S, Brunori G (eds) (2020) Innovation for sustainability. Research in rural sociology and development series, vol 25. Emerald Publishing Limited, Bradford
Kunneke R, Ménard C, Groenewegen J (2021) Network Infrastructures: technology meets Institutions. Cambridge Univer-
sity Press, Cambridge
Ménard C, Shirley M (2022) Advanced introduction to new institutional economics. Edward Elgar Publishing, Cheltenham
P bli h
’ N t Acknowledgements Acknowledgements
Thanks to Gianluca Brunori, Gaetano Martino and two referees for the very useful feedback. All opinions and errors are
mine. Disentangling institutions: a challenge (4) Last, meso-institutions function as
go-between, informing operators of the motivation behind the adoption of certain rules
and/or providing policy-makers feedback about the acceptability and feasibility of cer-
tain rules. There might be other functions fulfilled by meso-institutions. hi
Figure 1 summarizes the key role of the three institutional layers, suggests areas of
overlapping responsibilities, and indicates the need to introduce technology in the
model. For example, innovation (e.g., internet, or AI) may require changes in the rules
of the game, impair responsibilities of regulatory agency(ies), impact the organization of
transactions. To sum up, at least two major tasks are waiting researchers wishing to better under-
stand the nature, role, and impact of institutions on economic activities. One is to
develop further a theory of institutions that better integrate their different dimensions,
paying special attention to the meso-institutional layer. The other is to look for solutions
to the problem of measuring the impact of institutions on performance, which might as Page 3 of 3 Ménard Agricultural and Food Economics (2022) 10:16 Ménard Agricultural and Food Economics TECHNOLOGIES
MACRO-INSTITUTIONS
Institutions (e.g.,Parliaments, Constitutional
Courts) through which constitutive rules
(e.g., Common Agricultural Policy) are
established, rightsdefined and allocated. MESO-INSTITUTIONS
Institutional devices (e.g., regulatory agencies,
public bureaus) and mechanisms (e.g.,
protocols) transforming general rules and
norms into specific standards that frame the
playing field for operators and users. MICRO-INSTITUTIONS
Institutional arrangements (e.g., firms,
cooperatives) through which transactions
are organized (e.g., contracts) making rules
and norms operational
Fig. 1 Institutional layers, their overlapping areas and their interdependence with technology well go through a better understanding of the role of meso-institutions. These are excit-
ing tasks for a new generation of economists wishing to grasp the central role of institu-
tions in the running and dynamics of our economic systems. well go through a better understanding of the role of meso-institutions. These are excit-
ing tasks for a new generation of economists wishing to grasp the central role of institu-
tions in the running and dynamics of our economic systems. Publisher’s Note
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Using allocative efficiency analysis to inform health benefits package design for progressing towards Universal Health Coverage: Proof-of-concept studies in countries seeking decision support
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Background Countries are increasingly defining health benefits packages (HBPs) as a way of progress-
ing towards Universal Health Coverage (UHC). Resources for health are commonly con-
strained, so it is imperative to allocate funds as efficiently as possible. We conducted
allocative efficiency analyses using the Health Interventions Prioritization tool (HIPtool) to
estimate the cost and impact of potential HBPs in three countries. These analyses explore
the usefulness of allocative efficiency analysis and HIPtool in particular, in contributing to pri-
ority setting discussions. PLOS ONE PLOS ONE RESEARCH ARTICLE Abstract Citation: Fraser-Hurt N, Hou X, Wilkinson T, Duran
D, Abou Jaoude GJ, Skordis J, et al. (2021) Using
allocative efficiency analysis to inform health
benefits package design for progressing towards
Universal Health Coverage: Proof-of-concept
studies in countries seeking decision support. PLoS ONE 16(11): e0260247. https://doi.org/
10.1371/journal.pone.0260247 Editor: M. Mahmud Khan, University of Georgia,
UNITED STATES Editor: M. Mahmud Khan, University of Georgia,
UNITED STATES Received: May 11, 2021
Accepted: November 5, 2021
Published: November 29, 2021 Using allocative efficiency analysis to inform
health benefits package design for
progressing towards Universal Health
Coverage: Proof-of-concept studies in
countries seeking decision support Nicole Fraser-HurtID1, Xiaohui HouID1*, Thomas Wilkinson1, Denizhan DuranID1, Gerard
J. Abou JaoudeID2, Jolene Skordis2, Adanna ChukwumaID1, Christine Lao Pena1, Opope
O. Tshivuila Matala1, Marelize Gorgens1, David P. Wilson3 Nicole Fraser-HurtID1, Xiaohui HouID1*, Thomas Wilkinson1, Denizhan DuranID1, Gerard
J. Abou JaoudeID2, Jolene Skordis2, Adanna ChukwumaID1, Christine Lao Pena1, Opope
O. Tshivuila Matala1, Marelize Gorgens1, David P. Wilson3 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 The World Bank Group, Washington, DC, United States of America, 2 University College London Institute
for Global Health, London, United Kingdom, 3 Bill & Melinda Gates Foundation, Seattle, Washington, United
States of America * xhou@worldbank.org * xhou@worldbank.org Introduction Many countries now pursue some form of universal health coverage (UHC) policy objective,
aligning with local and international commitments including the achievement of the Sustain-
able Development Goals [1, 2]. UHC implies that all people have access to the services they
need, of sufficient quality to be effective, and without financial hardship. The services must be
safe and effective, including access to affordable essential medicines and vaccines [3]. All peo-
ple in need should be reached, including the most disadvantaged, and health service users
should have improved financial protection, leading to a reduction in financial hardship experi-
enced by households due to medical costs. Following the United Nations Sustainable Develop-
ment Summit in 2015, many countries have adopted policy frameworks on universal access to
essential health services and committed resources for the expansion of service delivery [4]. To
reach UHC goals, countries need to both commit more money to health and get more health
for the money [5, 6]. Country experiences demonstrate the importance of increased public
financing for health care through compulsory, prepaid revenues for making UHC progress
[7]. One key mechanism to address value for money is the development and provision of an
evidence-based health benefits package (HBP) [8]. Funding: The authors received no specific funding
for this specific work. Competing interests: The authors have declared
that no competing interests exist. An HBP, sometimes called an essential or basic package of health services, is a defined set of
services to be made available to everyone in the country [9]. Given that resources for health are
limited, priority setting is inevitable when developing HBPs and decisions must be made on
which interventions will be included. Priority setting for HBP design and progress toward
UHC can involve balancing trade-offs between different UHC dimensions, affordability, as
well as other locally relevant or valued criteria [7, 8, 10]. A common objective that guides HBP
design is the efficient use of resources to maximize population health outcomes [11]. To meet
this objective, a systematic and evidence-based consideration of the health gains achievable
from many potential interventions is required. The 3rd edition of Disease Control Priorities (DCP3) proposed an evidence-based set of
health interventions that are expected to provide good value for money in low- and middle-
income settings, address a significant disease burden, and are feasible to implement in a large
set of countries striving for UHC [12]. Methods and findings HIPtool is an open-access and open-source allocative efficiency modelling tool. It is pre-
loaded with publicly available data, including data on the 218 cost-effective interventions
comprising the Essential UHC package identified in the 3rd Edition of Disease Control Priori-
ties, and global burden of disease data from the Institute for Health Metrics and Evaluation. For these analyses, the data were adapted to the health systems of Armenia, Coˆte d’Ivoire
and Zimbabwe. Local data replaced global data where possible. Optimized resource alloca-
tions were then estimated using the optimization algorithm. In Armenia, optimized spending
on UHC interventions could avert 26% more disability-adjusted life years (DALYs), but even
highly cost-effective interventions are not funded without an increase in the current health
budget. In Coˆte d’Ivoire, surgical interventions, maternal and child health and health promo-
tion interventions are scaled up under optimized spending with an estimated 22% increase
in DALYs averted–mostly at the primary care level. In Zimbabwe, the estimated gain was
even higher at 49% of additional DALYs averted through optimized spending. Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0260247 Copyright: © 2021 Fraser-Hurt et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: The data underlying
the Armenia analysis are available in a databook on: Data Availability Statement: The data underlying
the Armenia analysis are available in a databook on: 1 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 PLOS ONE Allocative efficiency analysis to inform health benefits package https://dataverse.harvard.edu/dataset.xhtml? persistentId=doi:10.7910/DVN/LSX0BD Data for
Cote d’Ivoire and Zimbabwe largely came from
published government sources, as outlined in the
article. Access to the unpublished data is subject to
restrictions owing to privacy and ethics policies in
these countries. The data were made available to
the World Bank as technical partner in health
system analysis. Requests for access to the
specific unpublished data therefore need to be
made to the Ministry of Health and Child Care in
Zimbabwe and Ministry of Health and Public
Hygiene in Coˆte d’Ivoire. Conclusions HIPtool applications can assist discussions around spending prioritization, HBP design and
primary health care transformation. The analyses provided actionable policy recommenda-
tions regarding spending allocations across specific delivery platforms, disease programs
and interventions. Resource constraints exacerbated by the COVID-19 pandemic increase
the need for formal planning of resource allocation to maximize health benefits. Methods and findings Inquiries can be made to
the article’s corresponding author for further
guidance. The authors confirm that they did not
have special access privileges that others would
not have. PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Introduction The ‘model HBP’ recommended by DCP3, called Essen-
tial UHC (EUHC), is comprised of 218 interventions delivered through different platforms
considered representative of a typical health system: (a) population, (b) community, (c) health
centers, (d) first-level hospitals and (e) referral hospitals. For the most resource constrained
environments, DCP3 recommends the Highest Priority Package (HPP), which is a subset of
115 EUHC interventions selected based on a number of criteria [5]. The average costs and
mortality averted of the EUHC and HPP interventions were estimated by the DCP3 initiative,
which showed also that cost of implementation will vary by country context [5, 13, 14]. This is
because the individual demographic and epidemiological situations, delivery costs, available
budgets, and implementation structures vary from country to country. The DCP3 EUHC
package is intended as a starting point to help guide the development of an HBP or review PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 2 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package existing provision, and it can be supplemented by data from the Tufts Registry [15] and the
WHO UHC compendium [16]. While there is substantial global guidance for HBP design, countries must adjust recom-
mendations based on local contexts and constraints. The Health Interventions Prioritization
tool (HIPtool) has been developed to widen access to allocative efficiency analyses and assist
countries in selecting, synthesizing and translating global evidence and HBP recommenda-
tions to their own health system’s context. The tool, available as an online application (hiptool. org), uses a mathematical algorithm to determine allocations across defined health interven-
tions which provide optimal health impact. HIPtool analyses are intended to initiate or con-
tribute to the health resource allocation processes. To reduce the time and costs typically
associated with allocative efficiency analyses, HIPtool draws on publicly available data sources
including DCP3 health intervention data and the Institute for Health Metrics and Evaluation
(IHME) global burden of disease database. Publicly available data can be customized in the
tool to reflect country context, or replaced entirely with local data sources when available. The
data are used to estimate the cost and health impact, in terms of disability-adjusted life years
(DALY) averted, of various potential interventions and HBPs. The HIPtool optimization algo-
rithm also estimates an optimized resource allocation within defined resource envelopes. Introduction The
HIPtool is designed to assist decision makers in generating evidence to aid policy discussions
around health intervention prioritization and HBP design. The World Bank led early applications of HIPtool in three countries–Armenia, Coˆte d’Ivoire
and Zimbabwe (Table 1). These case studies are the basis for the analyses presented in this paper. This paper summarizes the experiences from HIPtool proof-of-concept studies in these
three countries. With the increased demand for and availability of mathematical modeling for
decision support in health, the paper aims to share the lessons learnt in the process. PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 PLOS ONE https://doi.org/10.1371/journal.pone.0260247.t001 efficiency, equity and financial risk protection, reduce the potential for political capture of pri-
ority setting, and inform how limited health resources could be more efficiently allocated to
progress towards UHC. PLOS ONE Table 1. Key data on the three country case studies. Indicator
Armenia
Coˆte d’Ivoire
Zimbabwe
Income classification
Upper middle-income
Lower middle-income
Lower middle-income
Region
Europe & Central Asia
Sub-Saharan Africa
Sub-Saharan Africa
Population size (2019)
2.97 million
25.72 million
14.65 million
Domestic government
health expenditure (% of
GDP, 2018)
1.24%
1.21%
1.32%
Health expenditure per
capita (current US$, 2018)
422.28
71.88
140.32
Domestic general
government health
expenditure per capita
(current US$, 2018)
52.11
20.71
39.25
Out of pocket expenditure
as % of health expenditure
(2018)
84.3%
39.4%
24.4%
UHC effective coverage
index
62.4
43.0
54.5
Top 3 risk factors driving
death and disability
combined (2019)
High blood pressure, tobacco, dietary risks
Malnutrition, air pollution, water-
sanitation-hygiene
Malnutrition, unsafe sex, air pollution
Outcomes:
Life expectancy at birth,
74.9 years
57.4 years
61.2 years
2018 (versus in 2000)
(71.4 in 2000)
(49.6 in 2000)
(44.6 in 2000)
Maternal mortality
ratio, 2017
26/100,000 live births
617/100,000 live births
458/100,000 live births
Under five mortality
rate, 2019
11.8 /1,000 live births
79.3 /1,000 live births
54.6 /1,000 live births
Health benefits package
explicitly defined
HBP since 1997. Covers primary health care
services for all, and other services including
hospital care and diagnostics for 30 socially
vulnerable and special groups. The per-
capita payment system aggregates services at
the PHC level within the HBP
Universal health insurance recently
launched with a benefits package under
development
The publicly financed system has the
National Health Services Package guiding
resource allocation
Efficiency and rationale
for HIPtool application
Efficiency has been gained through
reduction in excess hospital capacity, and
the HBP is being reviewed. The HIPtool
application and other analytical support
activities, led by the MOH with support
from development partners, aim to
contribute to a structured and systematic
approach to HBP re-design
An actuarial study projected an annual
financing gap of >$258 million in the new
health insurance, doubling by 2028, if
contribution and expenditure levels
remained stable. This pointed to the need
for an evidence-based prioritization
mechanism with supportive analytics such
as HIPtool use
Health financing analyses suggested that to
achieve better health outcomes, spending
efficiency should be improved. The HIPtool
application was conducted to help identify
areas or interventions that should be
prioritized in order to improve spending
efficiency Table 1. Key data on the three country case studies. Overall process HIPtool applications constitute part of a broader set of processes and stakeholder consultations
at country level, aiming to review public resource allocation, improve allocative efficiency, or
support HBP definition. The steps of HIPtool implementation, including what data are
required and expected model outputs, are shared with relevant stakeholders. Consensus is
built around data sources and usage, stakeholder roles, secondary analyses, budget levels to be
considered and optimization objectives. The tool facilitates a structured, evidence-driven dia-
logue focused on advancing UHC and defining a package of essential health interventions
which is optimized to improve allocative efficiency in the following ways: • HIPtool links data on intervention coverage, spending and cost-effectiveness to support dis-
cussions on what impact is being achieved with publicly financed interventions • The optimization outputs suggest changes that could improve the impact of a set of
interventions • The ability of HIPtool to optimize different budget envelopes, can frame discussions on fiscal
space for health and health financing • The tool’s options for weighting the relative importance of health impact, financial risk pro-
tection (FRP) and equity, contribute to discussions on how public service provision can pro-
vide value for money Overall, stakeholder engagement throughout the HIPtool implementation process can cre-
ate a space to discuss health reform options including tradeoffs between objectives of Overall, stakeholder engagement throughout the HIPtool implementation process can cre-
ate a space to discuss health reform options including tradeoffs between objectives of 3 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Allocative efficiency analysis to inform health benefits package HIPtool application A HIPtool application is designed to help answer the following questions, subject to available
data: 4 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 PLOS ONE Allocative efficiency analysis to inform health benefits package 1. What are the current health interventions that are being implemented, what is the current
level of spending on these interventions and what portion of disease burden does current
spending avert? 2. How can spending be best allocated across essential interventions, health delivery platforms
and disease programs to maximize DALYs averted, given the country’s scale-up targets and
intervention cost-effectiveness? 3. What health services or interventions outside of the optimized HBP would be cost-effective
and important to deliver? 4. How do changes in available funding affect the interventions included in an optimized
HBP and associated DALYs averted? 5. What are the gaps in data to enable effective priority setting across essential health
interventions? The default input parameters in HIPtool come from DCP3 [5], IHME’s burden of disease
study [17] and Tufts Cost Effectiveness Analysis Registry [15], among other sources. Imple-
mentation broadly follows five stages (Fig 1) and a detailed description of HIPtool is available
in S1 Appendix. The first stage maps a country’s existing HBP or publicly financed health
interventions to the pre-loaded EUHC interventions (see S2 Appendix). Several HBP services
delivered in a country might be grouped under one EUHC intervention, and a crosswalk
might reveal mismatches between the two taxonomies that can be individually resolved. The Fig 1. Overview of the five stages of the HIPtool application. Source: Authors’ summary. Note: DCP3 = Third edition of Disease Control Priorities,
UHC = Universal health coverage. https://doi org/10 1371/journal pone 0260247 g001 Fig 1. Overview of the five stages of the HIPtool application. Source: Authors’ summary. Note: DCP3 = Third edition of Disease Control Priorities,
UHC = Universal health coverage. https://doi.org/10.1371/journal.pone.0260247.g001 https://doi.org/10.1371/journal.pone.0260247.g001 5 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 PLOS ONE Allocative efficiency analysis to inform health benefits package selected interventions can either be grouped into 21 care packages as per DCP3 (S2 Appendix)
or the country’s own disease control programs. The pre-loaded information on care delivery
platforms (community, health center, first-level hospital, referral hospital, population-based)
can be edited based on the country’s health system. HIPtool application Some interventions are defined across mul-
tiple levels of care, for example child delivery interventions are at community (management of
low-risk labor symptoms and referral services), health center (basic emergency newborn and
obstetric care, BEmNOC), and hospital (comprehensive emergency newborn and obstetric
care, CEmNOC) levels of care. The second stage of HIPtool applications involves using data on current and target cover-
age. This process involves triangulation, estimation and judgement calls, as intervention cover-
age among those in need or eligible is often not reported. Strategic documents and plans as
well as key informants can inform scale-up coverage targets. Where unavailable, DCP3 cover-
age estimates and a target coverage of 80% are used [18]. Pre-loaded unit costs, defined as the
annual cost to deliver an intervention per person, can then be automatically adjusted for each
country using the DCP3 costing model online tool [18]. In general, local unit costs are used
where available, and the pre-loaded, adjusted unit costs are used to fill the gaps. The third stage uses data triangulation and mapping techniques to validate the bottom-up
costing amounts with reported expenditure levels, such as health spending in National Health
Accounts, adjusting cost parameters for inflation to the required year of analysis. The fourth stage of a HIPtool analysis begins by considering incremental cost-effectiveness
ratios (ICER) which are pre-loaded in the tool for most interventions based on DCP3 EUHC
data. Where the default EUHC ICERs provided from the DCP3 data are not considered appro-
priate for the local health system context, users can recalibrate ICERs by, for instance, substi-
tuting ICERs with more recent estimates from the literature or the continuously updated Tufts
Cost-Effective Analysis Registry [16]. Each intervention is also associated with FRP and equity
scores based on the DCP3 methodology [19]. Once ICER estimates are selected, a ‘quality
reduction’ factor is applied to reflect the fact that intervention effect sizes may be lower when
implemented at scale in the health system context than that reported in the literature. A default
30% reduction in intervention effect is applied in HIPtool, which can be adjusted by users
[20]. Intervention input data is then combined to estimate a ‘maximum potential impact’
(MPI) for each intervention, which is the estimated maximum impact an intervention could
have on the causes of the disease burden it is linked to. HIPtool application The MPI parameter defines the ceiling
of investment for each intervention during the optimization process, and is estimated assum-
ing a linear relationship between spending, cost-effectiveness, and current and target nominal
coverage (see S1 Appendix on how MPI is calculated). The fifth stage requires the setting of the budget level to be optimized and the optimization
objective. This is a user-defined combination of maximizing DALYs averted, equity scores and
FRP. HIPtool’s mathematical optimization function enables all the included health interven-
tions to be assessed simultaneously, enabling the “whole of package” assessment that can be
applied much faster than assessing individual interventions manually. The optimization mod-
ule of the HIPtool is detailed in S1 Appendix. Model outputs on optimized spending patterns
and health impact require extensive review and iterative analysis reflecting policy-relevant
questions. Results There were variations in how HIPtool was implemented in the three countries. There were
also variations in the results and insights that could be gained at this proof-of-concept stage. We present them hereunder in brief (detailed technical country reports are in preparation or 6 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 PLOS ONE Allocative efficiency analysis to inform health benefits package published [21]). The calculations required for a country application can be viewed in a data
book on the link http://hdl.handle.net/10986/35347 (Armenia example). Armenia: Rich data environment allows localization of HIPtool and
provides policy-relevant outputs Epidemiological or demographic estimates were scaled down
based on assumptions about the proportion of individuals requiring the intervention in a sin-
gle year. For instance, diabetes screening in the target group is recommended to be done every
three years, so the number of undiagnosed individuals in the eligible age range was divided by
three to estimate the number requiring screening in 2019. Data were sourced from demo-
graphic and key population estimates, Armenia’s vaccination schedule, disease burden data
from IHME, national surveys [22–25] and other sources. Unit costs were calculated by sum-
ming all claims in the ArMed e-health system divided by the total number of claims in 2019,
for all HBP services grouped under a specific AUHC intervention. Spends in four per-capita
HBP codes were apportioned to nine PHC-level AUHC interventions using estimated target
population, intervention coverage and unit cost. Similarly, spends for drug provision (two
HBP codes for adults, two for children) were apportioned to a total of 15 AUHC interventions. MOH central-level spend for vaccines was mapped to vaccination-related AUHC
interventions. 2. Intervention coverage, target groups and unit costs. This step replaced pre-loaded data
with Armenia-specific data. For each AUHC intervention, 2019 coverage in the target popula-
tion was estimated. The population ‘in need’ was defined using estimates on incidence or prev-
alence. The population ‘eligible’ drew on guidelines and demographic data (e.g. birth cohort,
women of reproductive age). Epidemiological or demographic estimates were scaled down
based on assumptions about the proportion of individuals requiring the intervention in a sin-
gle year. For instance, diabetes screening in the target group is recommended to be done every
three years, so the number of undiagnosed individuals in the eligible age range was divided by
three to estimate the number requiring screening in 2019. Data were sourced from demo-
graphic and key population estimates, Armenia’s vaccination schedule, disease burden data
from IHME, national surveys [22–25] and other sources. Unit costs were calculated by sum-
ming all claims in the ArMed e-health system divided by the total number of claims in 2019,
for all HBP services grouped under a specific AUHC intervention. Spends in four per-capita
HBP codes were apportioned to nine PHC-level AUHC interventions using estimated target
population, intervention coverage and unit cost. Similarly, spends for drug provision (two
HBP codes for adults, two for children) were apportioned to a total of 15 AUHC interventions. Armenia: Rich data environment allows localization of HIPtool and
provides policy-relevant outputs Policy context. The HIPtool application was embedded in multiple other studies, includ-
ing an actuarial costing of a benefits package, an assessment of strategic purchasing in the
health sector and a projection of revenues from tax- and non-tax sources. All the studies
focused on providing technical support for UHC and benefit package re-design in Armenia. The model was implemented in the first half of 2020 using 2019 HBP data and National Health
Accounts (NHA) data up to and including 2018. The process was an inclusive one in which
HIPtool Focal Points from various Ministry of Health (MOH) units, the State Health Agency,
National Institute of Health, and World Bank Project Implementation Unit were instrumental
in making data available in formats usable in the model. Modelling scenarios were discussed,
including a higher budget scenario using a 38% increase in the current health budget level
(associated with a hypothetical increase of the state budget to health from 5.8% to 8.0%). Focal
Points were briefed about the tool and acted as key resource persons during the entire study to
ensure appropriate data use and review. The State Health Agency collaborated closely by
extracting large data sets from its ArMed e-health system. This system includes data on
approximately 3,700 HBP service codes, which can be specific to defined social groups, geo-
graphical areas and refund entitlement levels. Multiple HBP service codes relate to per-capita
services provided for free at primary health care (PHC) level pertaining to general medical
practice, pediatrics, obstetrics/ gynecology and family medicine. Key takeaways and results from HIPtool implementation in Armenia. 1. Intervention
list. The mapping and matching of Armenia’s publicly financed health interventions to the
pre-loaded EUHC interventions resulted in 135 Armenia UHC (AUHC) interventions which
could enter the optimization analysis in the HIPtool. The step involved the review of approxi-
mately 3,700 coded HBP services and grouping them under EUHC interventions. The 135
retained interventions belonged to 19 of the DCP3’s 21 care packages (excluding neglected
tropical diseases and environmental health packages). p
p
g
2. Intervention coverage, target groups and unit costs. This step replaced pre-loaded data
with Armenia-specific data. For each AUHC intervention, 2019 coverage in the target popula-
tion was estimated. The population ‘in need’ was defined using estimates on incidence or prev-
alence. The population ‘eligible’ drew on guidelines and demographic data (e.g. birth cohort,
women of reproductive age). Armenia: Rich data environment allows localization of HIPtool and
provides policy-relevant outputs MOH central-level spend for vaccines was mapped to vaccination-related AUHC
interventions. 7 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 PLOS ONE Allocative efficiency analysis to inform health benefits package 3. Current spending. The analysis included government and Global Fund health spending. The 135 AUHC interventions were associated with 56% of total government health expendi-
ture as per the NHA (USD 93.809 million, of which 18.5% is within the capitation system). Another 44% of government health expenditure remained outside of AUHC interventions as
the respective services could not be mapped to any of the HIPtool interventions, or the spend
was linked to health worker salaries, infrastructure or consumables. Data triangulation across
reported spending levels was imperfect as the analysis used 2019 HBP spending but 2018 NHA
data, however, long-term NHA data was used to understand annual government health spend-
ing trends. Global Fund spending on AUHC interventions on HIV and TB was included in
the optimization (leading to a total amount of USD 95.653 million in the optimization). 4. Maximum potential impact. This was calculated by projecting the intervention coverage
levels in the HBP-eligible target groups to the Armenia population. Assumptions and judge-
ment calls had to be made during this part of the process. For example, when an HBP service
was provided for socially vulnerable and other special groups and emergency cases (e.g. surgi-
cal management of rectovaginal fistula), or socially vulnerable and other special groups, and
children<18 (e.g. proctologic and reconstructive colon surgeries). Again, triangulation of dis-
ease burden, HBP claims, eligibility and survey/statistical data was essential to develop best
possible estimates of population-level coverage. The tool’s pre-loaded ICER values were gener-
ally used but reviewed and selectively replaced with more appropriate values from the litera-
ture, especially where ICERs were relatively low compared to the disease burden and reported
spending. The pre-loaded FRP and equity scores were replaced by local scores, with highest
scores given to the most expensive AUHC interventions and those targeting the most vulnera-
ble groups (detailed in S3 Appendix). 5. Optimization and interpretation of results. Modeling scenarios varied several parameters
to explore outcomes, including i) Budget: 2019 budget or a 38% higher budget; ii) Optimiza-
tion constraints: Only 10% allowable coverage increase, or unconstrained; iii) Optimization
weights for DALYs, FRP and equity: Equal, or high DALY weight, or low equity weight. Armenia: Rich data environment allows localization of HIPtool and
provides policy-relevant outputs The
model outputs were overall similar for different weights given to DALYs, FRP and equity in
the optimization step. Outputs were aggregated by each of the 21 EUHC care packages but not
by service delivery platform due to many interventions using multiple platforms. It was esti-
mated that the 135 interventions at 2019 budget and coverage averted 120,000 DALYs, and
that optimized spending could increase the impact to 151,000 DALYs (Fig 2). If the budget
available to fund the 135 essential AUHC interventions could be increased to the hypotheti-
cally feasible level (i.e. 38% increase on the 2019 health budget), an estimated 166,000 DALYs
could be averted by the included interventions compared to the actual 2019 funding level and
allocations. Coˆte d’Ivoire: Model implementation using pre-loaded DCP3 data provides
directional results for investment in disease programs Key data sources were the population census [28], the Health Management Informa-
tion System, the 2016 Multiple Indicator Cluster Survey [29] and IHME prevalence estimates. 3. Current spending. The analysis included all health spending irrespective of financing
source, including out of pocket expenditures. Spending was calculated by multiplying the unit
costs, target population and target coverage levels. A total of US$ 1.433 billion was included in
the optimization and spending shares across disease programs was compared to 2016 NHA
program spending data for validation. Significant mismatches were seen for NCD and surgical
interventions, which constituted about a quarter of national health expenditures, but the NHA
did not have detailed expenditure data at the intervention level which is needed for HIPtool
optimization. In the final model, NCDs, malaria, maternal health and HIV were the disease
programs with the largest expenditures adding up to almost 70% of all health spending, which
was in line with the 2016 NHA spending patterns. 3. Current spending. The analysis included all health spending irrespective of financing
source, including out of pocket expenditures. Spending was calculated by multiplying the unit
costs, target population and target coverage levels. A total of US$ 1.433 billion was included in
the optimization and spending shares across disease programs was compared to 2016 NHA
program spending data for validation. Significant mismatches were seen for NCD and surgical
interventions, which constituted about a quarter of national health expenditures, but the NHA
did not have detailed expenditure data at the intervention level which is needed for HIPtool
optimization. In the final model, NCDs, malaria, maternal health and HIV were the disease
programs with the largest expenditures adding up to almost 70% of all health spending, which
was in line with the 2016 NHA spending patterns. 4. Maximum potential impact. This step linked the 179 interventions to the pre-loaded
ICER values and reviewed the relationship between the burden of disease addressed by an
intervention, as well as intervention spending and impact. The default 30% ‘quality reduction’
factor was applied, and the pre-loaded DCP3 data on intervention FRP and equity scores were
maintained. 5. Optimization and interpretation of results. The optimization was run using target cover-
age levels. For interventions with coverage above 80%, target coverage was kept equal. For
communicable disease interventions with coverage below 80%, target coverage was fixed at
80% following stakeholder discussions. Coˆte d’Ivoire: Model implementation using pre-loaded DCP3 data provides
directional results for investment in disease programs The
Directorate of Health Services overseeing the individual disease programs was also involved
and provided essential intervention data. The Ministry of Health team also regularly liaised
with the Directorate of Budget in terms of the results and how they could potentially be used
to inform decision-making processes. Evaluation, the newly launched health insurance agency, and health service directorates. The
Directorate of Health Services overseeing the individual disease programs was also involved
and provided essential intervention data. The Ministry of Health team also regularly liaised
with the Directorate of Budget in terms of the results and how they could potentially be used
to inform decision-making processes. Key takeaways and results from HIPtool implementation in Coˆte d’Ivoire. 1. Interven-
tion list. Mapping existing services to the 218 EUHC interventions in HIPtool resulted in 179
interventions retained for the Coˆte d’Ivoire study. Given the absence of a single national HBP,
stakeholder consultations and document review helped to arrive at the intervention list. 2. Intervention coverage, target groups and unit costs. For each intervention, coverage and
target group data were collected considering levels of care. The tool’s pre-loaded data were
used for the interventions’ default coverage levels and unit cost, unless the 2016 OneHealth
assessment [26] the 2016 Multiple Indicator Cluster Survey [27] or other national data could
be used. Only 26% of the interventions had data on local target populations and intervention
coverage. Data on non-communicable diseases (NCDs) were especially scarce and interna-
tional estimates were used. All pre-loaded unit cost data used were adjusted automatically in
HIPtool. Key data sources were the population census [28], the Health Management Informa-
tion System, the 2016 Multiple Indicator Cluster Survey [29] and IHME prevalence estimates. 2. Intervention coverage, target groups and unit costs. For each intervention, coverage and
target group data were collected considering levels of care. The tool’s pre-loaded data were
used for the interventions’ default coverage levels and unit cost, unless the 2016 OneHealth
assessment [26] the 2016 Multiple Indicator Cluster Survey [27] or other national data could
be used. Only 26% of the interventions had data on local target populations and intervention
coverage. Data on non-communicable diseases (NCDs) were especially scarce and interna-
tional estimates were used. All pre-loaded unit cost data used were adjusted automatically in
HIPtool. Coˆte d’Ivoire: Model implementation using pre-loaded DCP3 data provides
directional results for investment in disease programs Policy context. HIPtool was implemented in Coˆte d’Ivoire between May 2018-October
2019, which overlapped with discussions in the country regarding ambitious health sector
reforms. Reforms under discussion included the launch of a national health insurance scheme,
the scale up of strategic purchasing and preparation of the Global Financing Facility (GFF)
investment case to strengthen the health system and improve maternal and newborn health
outcomes. Key national data came from the 2016 NHA and previous OneHealth Tool costing
exercises (internal government documents, unpublished). The World Bank team worked
closely with a Ministry of Health Core Team including representatives from the Cabinet (chief
health financing advisor to the Minister of Health), Finance, Planning, Monitoring and PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 8 / 21 PLOS ONE
Allocative efficiency analysis to inform health benefits package
PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021
9 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 9 / 21 Fig 2. Armenia model outputs on optimized resource allocation and health impact by EUHC care package (2019 vs. higher budget scenario). A) Estimated
spending. B) Estimated DALY impact. Source: Armenia HIPtool analysis. Note: Maximum allowable coverage increase = 90% (if 2019 baseline <90%, 95% (if
baseline 90–94%, 100% (if baseline 95+%); child delivery interventions fixed at 2019 levels; Equal weights for DALYs, FRP and equity. LOS ONE
Allocative efficiency analysis to inform health benefits package PLOS ONE Allocative efficiency analysis to inform health benefits package Fig 2. Armenia model outputs on optimized resource allocation and health impact by EUHC care package (2019 vs. higher budget scenario). A) Estimated
spending. B) Estimated DALY impact. Source: Armenia HIPtool analysis. Note: Maximum allowable coverage increase = 90% (if 2019 baseline <90%, 95% (if
baseline 90–94%, 100% (if baseline 95+%); child delivery interventions fixed at 2019 levels; Equal weights for DALYs, FRP and equity. Fig 2. Armenia model outputs on optimized resource allocation and health impact by EUHC care package (2019 vs. higher budget scenario). A) Estimated
spending. B) Estimated DALY impact. Source: Armenia HIPtool analysis. Note: Maximum allowable coverage increase = 90% (if 2019 baseline <90%, 95% (if
baseline 90–94%, 100% (if baseline 95+%); child delivery interventions fixed at 2019 levels; Equal weights for DALYs, FRP and equity. https://doi.org/10.1371/journal.pone.0260247.g002 Evaluation, the newly launched health insurance agency, and health service directorates. PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Coˆte d’Ivoire: Model implementation using pre-loaded DCP3 data provides
directional results for investment in disease programs For NCDs, target coverage was fixed at 30% following
stakeholder discussions, due to very low baseline levels of provision. Optimization results were
presented by care delivery platform (Fig 3) and national disease programs (S4 Appendix)
rather than DCP3’s care packages, to ensure relevance to existing planning and budget lines. The community platform gained most spending in the optimization (+68%), followed by the
population-based platform (+31%) and the referral and specialist hospitals (+19%) (Fig 3A). Despite a 12% reduction in spending for each the health centre and the first level hospital plat-
forms, the estimated DALY impacts were positive for all five platforms (Fig 3B). Shifts in
spending for care delivery platforms ranged from a 68% increase for community-based PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 10 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package interventions to a 12% decrease for Health Centre and first level hospital. Under optimized
spending scenarios, surgery, maternal health, child health and health promotion interventions
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Fig 3. Coˆte d’Ivoire model outputs on actual and optimized 2016 spending and impact by care delivery platform. A) Estimated spending. B) Estimated DALY
impact. Source: Coˆte d’Ivoire HIPtool analysis. https://doi.org/10.1371/journal.pone.0260247.g003 Fig 3. Coˆte d’Ivoire model outputs on actual and optimized 2016 spending and impact by care delivery platform. A) Estimated spending. B) Estimated DALY
impact. Source: Coˆte d’Ivoire HIPtool analysis. Fig 3. Coˆte d’Ivoire model outputs on actual and optimized 2016 spending and impact by care delivery platform. A) Estimated spending. B) Estimated DALY
impact. Source: Coˆte d’Ivoire HIPtool analysis. htt
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0260247 003 Fig 3. Coˆte d’Ivoire model outputs on actual and optimized 2016 spending and impact by care delivery platform. A) Estimated spending. B) Estimated DALY
impact Source: Coˆte d’Ivoire HIPtool analysis Fig 3. Coˆte d’Ivoire model outputs on actual and optimized 2016 spending and impact by care delivery platform. A) Estimated spending. B) Estimated DALY
impact. Source: Coˆte d’Ivoire HIPtool analysis. https://doi.org/10.1371/journal.pone.0260247.g003 https://doi.org/10.1371/journal.pone.0260247.g003 interventions to a 12% decrease for Health Centre and first level hospital. Under optimized
spending scenarios, surgery, maternal health, child health and health promotion interventions
were scaled up significantly. The DALYs averted increased for all disease programs due to a
change in interventions included, from 9.1 million DALYs averted by current spending, to
11.1 million DALYs averted by the optimized allocation. Coˆte d’Ivoire: Model implementation using pre-loaded DCP3 data provides
directional results for investment in disease programs Optimization significantly increased
impact at the primary care level (i.e., community and health center), particularly for preventive PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 11 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package interventions in the community, going from 3.96 million DALYs to 4.82 million DALYs
averted at the health center level, and going from 3.34 million DALYs to 3.98 million DALYs
averted at the community level. Under the optimized scenario, although only 16% of the bud-
get is disbursed at the community level, this spend averts 36% of DALYs. Overall, optimized
spending at the community and primary care levels averted 79% of DALYs, for 45% of the
budgeted spend. Analysis of interventions with the largest spending increases and DALY
impact upon optimization (S4 Appendix) showed that four of the five interventions which, fol-
lowing optimization, see the most significant increase in DALYs averted are communicable
disease-related, followed by care for fractures. Zimbabwe: Extensive data integration in HIPtool enables model
implementation for decision support Policy context. In Zimbabwe, HIPtool was implemented during 2018–19 in response to
several health financing studies that pointed to an urgent need to improve spending efficiency
in Zimbabwe [30–33]. HIPtool implementation was guided and informed by interviews with
key officials from the Ministry of Health and Child Care (MHCC), the Ministry of Finance
and Economic Development, the Clinton Health Access Initiative and the World Health Orga-
nization, among other stakeholders. The analysis was based on 2016 fiscal year data, with
expenditure, cost and budget data extracted from the 2016 Resource Mapping Report [34] the
NHA Report [33] the National Health Strategy 2015–2020 [34] and MHCC’s 2016 expenditure
and appropriation account (unpublished). Also consulted were the 2015 National Health Ser-
vice (NHS) report (unpublished), WHO Global Health Observatory [35], Demographic and
Health Surveys [36], Multiple Indicator Cluster Surveys [37], UNAIDS HIV data [38], pub-
lished, peer-reviewed and grey literature, agencies’ databases including UN Populations Divi-
sion and UNICEF, and the Global Burden of Disease database for disease prevalence [19]. Key takeaways and results from HIPtool implementation in Zimbabwe. 1. Intervention
list. Most of the NHS package was successfully mapped to 176 of the 218 EUHC interventions
pre-loaded in HIPtool, using NHS protocols and consulting with local experts. 2. Intervention coverage, target groups and unit costs. Like the other country applications,
included interventions were linked to populations in need or eligible, coverage levels and unit
cost. Data was drawn from a broad range of sources and pre-populated HIPtool data was used
whenever required. Local unit costs from the National Health Strategy 2015–2020 mostly
related to MCH interventions. The resource mapping study provided data on HIV and malaria
spending. Otherwise, the pre-populated EUHC intervention unit costs were used, following
adjustment, and inflation to 2016. 3. Current spending. Intervention spending was estimated by combining unit cost and
annual utilization estimates for each of the interventions. Available expenditure and budget
data were then used to compare and validate aggregated intervention expenditure estimated
with HIPtool [39]. Of the estimated US$980 million spent on health from domestic govern-
ment and external sources, US$672 million (69%) were included in the optimization. The
remaining US$308 million were not included in the optimization, primarily because no direct
link could be established between spending and burden of disease. PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Zimbabwe: Extensive data integration in HIPtool enables model
implementation for decision support This applied for instance to
health systems strengthening, palliative care, prevention and relief of refractory suffering,
resuscitation with basic measures and NCD behavior change communication. Therefore, for
the 19 interventions or expenditure activities that were not included in the optimization, asso-
ciated spending was fixed and remained unchanged in the analysis. 3. Current spending. Intervention spending was estimated by combining unit cost and
annual utilization estimates for each of the interventions. Available expenditure and budget
data were then used to compare and validate aggregated intervention expenditure estimated
with HIPtool [39]. Of the estimated US$980 million spent on health from domestic govern-
ment and external sources, US$672 million (69%) were included in the optimization. The
remaining US$308 million were not included in the optimization, primarily because no direct
link could be established between spending and burden of disease. This applied for instance to
health systems strengthening, palliative care, prevention and relief of refractory suffering,
resuscitation with basic measures and NCD behavior change communication. Therefore, for
the 19 interventions or expenditure activities that were not included in the optimization, asso-
ciated spending was fixed and remained unchanged in the analysis. 4. Maximum potential impact. The interventions included in the optimization were linked
to the pre-loaded ICER values, but the team also consulted additional DCP3 data to find the PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 12 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package Fig 4. Zimbabwe model outputs on actual and optimized 2016 spending and impact by intervention. CB = Community-based, HC = Health Center, FLH = First
Level Hospital, RH = Referral and Specialty Hospital. A) Highest expenditure interventions (2016 actual versus optimized). B) Most impactful interventions (2016 actual
versus optimized). Source: Zimbabwe HIPtool analysis. Fig 4. Zimbabwe model outputs on actual and optimized 2016 spending and impact by intervention. CB = Community-based, HC = Health Center, FLH = First
Level Hospital, RH = Referral and Specialty Hospital. A) Highest expenditure interventions (2016 actual versus optimized). B) Most impactful interventions (2016 actual
versus optimized). Source: Zimbabwe HIPtool analysis. Fig 4. Zimbabwe model outputs on actual and optimized 2016 spending and impact by intervention. CB = Community-based, HC = Health Center, FLH = First
Level Hospital, RH = Referral and Specialty Hospital. A) Highest expenditure interventions (2016 actual versus optimized). B) Most impactful interventions (2016 actual
versus optimized). Source: Zimbabwe HIPtool analysis. Zimbabwe: Extensive data integration in HIPtool enables model
implementation for decision support https://doi.org/10.1371/journal.pone.0260247.g004 most appropriate values where ranges of ICERs from different contexts were available. All
ICERs were adjusted by the default 30% ‘quality reduction’ factor. most appropriate values where ranges of ICERs from different conte
ICERs were adjusted by the default 30% ‘quality reduction’ factor. 5. Optimization and interpretation of results. Full weighting was assigned to health maximi-
zation, prioritizing ICER values over the FRP and equity scores of the included interventions. Results from the HIPtool optimization analysis suggested that several interventions should be
allocated more funding, with the largest increases in the 2016 budget level allocated to inte-
grated community case management, HIV and STI testing, cotrimoxazole for children and
medical male circumcision (Fig 4A). Maternal and child health interventions remained critical
in the optimized allocation and most priority interventions were delivered at the community
and primary health center level. TB interventions remained a strong area of focus, involving
diagnosis and first-line treatment at the health center level and MDR-TB diagnosis and 5. Optimization and interpretation of results. Full weighting was assigned to health maximi-
zation, prioritizing ICER values over the FRP and equity scores of the included interventions. Results from the HIPtool optimization analysis suggested that several interventions should be
allocated more funding, with the largest increases in the 2016 budget level allocated to inte-
grated community case management, HIV and STI testing, cotrimoxazole for children and
medical male circumcision (Fig 4A). Maternal and child health interventions remained critical
in the optimized allocation and most priority interventions were delivered at the community
and primary health center level. TB interventions remained a strong area of focus, involving
diagnosis and first-line treatment at the health center level and MDR-TB diagnosis and 5. Optimization and interpretation of results. Full weighting was assigned to health maximi-
zation, prioritizing ICER values over the FRP and equity scores of the included interventions. Results from the HIPtool optimization analysis suggested that several interventions should be
allocated more funding, with the largest increases in the 2016 budget level allocated to inte-
grated community case management, HIV and STI testing, cotrimoxazole for children and
medical male circumcision (Fig 4A). Maternal and child health interventions remained critical
in the optimized allocation and most priority interventions were delivered at the community
and primary health center level. https://doi.org/10.1371/journal.pone.0260247.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Discussion There are substantial data on current and future trends in countries’ disease burdens, health
service costs and coverage, and the expected impact of specific health interventions. Decision
makers in health have an unprecedented body of evidence to draw on but navigating such
complex data can be challenging. HIPtool was developed to address this challenge by combin-
ing available data on intervention cost, coverage and effectiveness across all major disease pro-
grams of a health system, within a mathematical optimization algorithm. Mathematical
optimization tools use defined algorithms to process multiple data points to identify highest
(or lowest) values across multiple objectives within a defined constraint. Applied to HBP
design, mathematical optimization approaches can identify an “optimal” series of health inter-
ventions within a health budget constraint that will independently or jointly maximize mutu-
ally exclusive objectives such as health, equity, and financial risk protection. While the outputs of the HIPtool cannot answer all the questions about how to attain UHC
or how to formulate the specifics of an HBP, the results presented here demonstrate how a
model like HIPtool can provide directional results on high-impact, priority interventions and
programs in different settings and therefore indicate allocatively efficient investments. One strength of applying a model like HIPtool is that it can provide the structure for a sys-
tematic, evidence-based and transparent process that includes stakeholder engagement, review
and appraisal of the evidence and extensive discussion of policy options. All three proof-of-
concept studies demonstrated that the application of HIPtool informed in-country policy dis-
cussions on defining HBPs and identifying key spending priorities to bridge gaps to UHC and
support primary health care transformation. As such, HIPtool can be a key input to delibera-
tive and data-driven priority setting processes as countries define or update their benefits
packages. In Armenia, HIPtool could be localized to a large extent thanks to the richness of recent
local data. Tool implementation drove secondary analysis of HBP claims data and generated
‘unit prices’ for each AUHC intervention. The first ever health system-wide estimation of cov-
erage levels highlighted gaps of concern. With a few exceptions like diabetes treatment, it
highlighted insufficient public financing of services addressing chronic diseases and condi-
tions. Zimbabwe: Extensive data integration in HIPtool enables model
implementation for decision support TB interventions remained a strong area of focus, involving
diagnosis and first-line treatment at the health center level and MDR-TB diagnosis and PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 13 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package treatment at a first-level hospital. None of the interventions for which additional spending was
prioritized were at the referral and specialty hospital level or population-wide. The model esti-
mated that 938,000 DALYs could be averted through optimized allocations in addition to the
estimated 1.9 million DALYs averted by 2016 spending (Fig 4B). Under optimized spending,
health center interventions would account for 55% of all DALYs averted, and a further 583,000
(20%) and 366,000 (13%) DALYs could be averted through community and first-level hospital
interventions, respectively (S4 Appendix). The impact of referral and specialty hospital inter-
ventions decreases by 9,000 DALYs under optimized allocations of spending. The optimized
allocation of 2016 national health spending is therefore estimated to increase the impact of all
but one platform of care, and generate cost-savings through first-level hospital interventions
that yield greater impact with a 25% spending reduction. PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Discussion A more efficient HBP would provide better cover for conditions linked to old age, given
the rapidly aging population with high rates of Alzheimer’s disease, falls, hearing loss, cataract,
cancers, low back pain, and type 2 diabetes [40]. Given the high burden of ischemic heart dis-
ease and stroke, it was no surprise that optimization pointed to cardiovascular disease preven-
tion and management as a consistent top priority for funding. The significant increased
investment in musculoskeletal and cancer packages in the optimized allocation suggests there
are currently missed opportunities for health impact. The analysis identified specific areas to PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 14 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package increase value for money in the HBP, such as better cover for physiotherapy, long term man-
agement of heart and vascular diseases, primary prevention of osteoporosis, palliative care,
HPV vaccination for schoolgirls, diagnosis and treatment of colorectal cancer, and several
rehabilitation interventions. However, it also demonstrated the limited impact of any HBP
with highly constrained levels of public funding. In Coˆte d’Ivoire, HIPtool implementation served as an entry point for priority setting dis-
cussions around the benefits package and available health interventions and has been wel-
comed by policymakers at both health and finance ministries. As there was no defined benefits
package in Coˆte d’Ivoire at the time of analysis and dissemination, the implementation of HIP-
tool also informed discussions on the ground pertaining to evidence-based mechanisms to
define and update the benefits package at different levels of care. Actionable policy implica-
tions included the need to increase spending on maternal health, child health and surgical
interventions, to reallocate resources within disease programs towards more cost-effective
interventions, and to increase spending on community interventions, particularly for NCDs,
due to the significant potential to avert DALYs. The insights from the process have contributed
to discussions on budgeting, the new health sector strategic plan and GFF investment case pro-
cesses, as well as building demand for routine data, especially for NCDs. Findings from the
HIPtool analysis also have implications for the health insurance and strategic purchasing
arrangements, as well as donor coordination and reallocation of donor budgets. The study
marks the beginning of possible longer-term technical and political engagement to improve
the allocative efficiency of health spending in Coˆte d’Ivoire. Discussion In Zimbabwe, the use of HIPtool, combined with other technical analyses, helped shape the
policy dialogue on how allocative efficiency can be increased in the health sector while bearing
in mind local economic, political and implementation realities. The model provided further
evidence of the high impact that well-funded HIV and TB treatment programs have, but also
showed the large share of health spending for HIV/AIDS, and the need to assess the allocative
efficiency of individual HIV interventions given the low resource levels for other priority pro-
grams. Consistent with global literature [41], the most impactful and prioritized interventions
were those delivered at lower platforms of care, such as the Primary Health Centers (PHCs). Given that spending on MCH and NCD-related interventions increased under an optimized
allocation, an emphasis on integrated care emerged as an important step to improving spend-
ing efficiency, such as integrated community case management and BEmNOC at PHCs. A
cost-effective HBP would shift public spending from hospitals to community and PHC plat-
forms of care, with community health workers and PHC staff spearheading integrated care
models. Similarly, NCD interventions could be more broadly delivered at the PHC and com-
munity levels to improve cost-effectiveness. Zimbabwe has identified strengthening budget
formulation processes as a key and urgent reform. The HIPtool implementation process repre-
sented an important step toward better use of local and international evidence for resource
allocation across disease programs. This is especially important in the context of the current
fiscal situation. In Zimbabwe, the use of HIPtool, combined with other technical analyses, helped shape the
policy dialogue on how allocative efficiency can be increased in the health sector while bearing
in mind local economic, political and implementation realities. The model provided further
evidence of the high impact that well-funded HIV and TB treatment programs have, but also
showed the large share of health spending for HIV/AIDS, and the need to assess the allocative
efficiency of individual HIV interventions given the low resource levels for other priority pro-
grams. Consistent with global literature [41], the most impactful and prioritized interventions
were those delivered at lower platforms of care, such as the Primary Health Centers (PHCs). HIPtool can be flexible in defining the total budget to be allocated, which can be tailored to
the country context. PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Limitations The studies had several limitations: First, since HIPtool adopts a health system perspective, it
is unable to capture cross-sectoral benefits that lie outside of the health budget. For example,
effects such as gains in productivity or school attendance cannot be captured. This means that
the positive impact of an HBP, as estimated by HIPtool, is likely to be an underestimate of the
full cross-sectoral impact. Second, since there is a trade-off between usability and flexibility,
there are many highly complex interactions between diseases and health interventions that
cannot be captured in full, as these would require more data than are available in most country
contexts. As a consequence, overlaps and synergies between the interventions included in HIP-
tool are not considered, and must be accounted for in the data being uploaded or parameteri-
sation of intervention MPIs. Also, only the static first-order impacts are currently
incorporated into the analyses. Third, HIPtool is not a disease modelling tool and therefore
does not currently account for disease progression and infectiousness, which would introduce
substantial complexity and data needs. Instead, it builds on the best existing projections of dis-
ease burden and studies of intervention effects in terms of mortality and DALYs averted. Fourth, the tool does not provide a timeline for intervention scale-up, which is instead
explored by running multiple scenarios. As such the analyses did not include a dynamic tem-
poral dimension or adjustments in effectiveness or cost at scale, but rather emphasize the pos-
sibility of reallocating resources at any given point. Fifth, as with any mathematical model,
there are numerous data limitations. For instance, the demographic data might not reflect true
population sizes in a country due to migration which is for instance significant among men in
Armenia. The analyses performed by HIPtool are only as valid as the data entered into it. How-
ever, HIPtool revealed important data gaps and “made a case” for increased focus and invest-
ment in the collection of data supporting decision-making. Moving forward, the UHC
compendium [43] recently released by WHO provides a comprehensive database of interven-
tions and associated data that can be used as inputs in HIPtool and address a number of exist-
ing data challenges. The studies had several limitations: First, since HIPtool adopts a health system perspective, it
is unable to capture cross-sectoral benefits that lie outside of the health budget. Discussion In both Zimbabwe and Coˆte d’Ivoire, development partner financing is a
significant source of overall health financing, thus the interventions prioritization was ana-
lyzed including both public and development partner financing in health. The tool is also effi-
cient in calculating and comparing various budget scenario analyses, normally within a few
minutes once the underlying data are complete and validated, to assist health policy makers in
exploring the impact of budget change and trade-offs across averted DALYs, equity and FRP. Through all three applications of HIPtool, useful lessons were learnt about the importance
of stakeholder collaboration during tool implementation. Engaging stakeholders early and PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 15 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package often, identifying a champion, seeking consensus, and joint data identification and vetting were
all essential to a well-supported process. Ad-hoc consultation with data owners and the securing
of expert support for customized analyses helped ensure the quality of model inputs. Stakehold-
ers’ interest in capacity building on allocative efficiency modeling and in future tool use signaled
an understanding of the important role analytics can play in attaining value-for money HBPs
and UHC goals. This understanding and institutional knowledge gained through these HIPtool
applications has the potential to improve the quality and speed of future analyses. HIPtool offers an interactive way of engaging with stakeholders. Once the tool data is
adjusted and deemed appropriate, users can test policy scenarios and update model inputs
continuously. In Coˆte d’Ivoire and Zimbabwe, a significant value of this proof-of-concept
application was that discussions on benefits packages started taking place for the first time. National strategic plans in both countries tend to be designed and executed in a disease-spe-
cific manner, without sector-wide prioritization. These discussions, if continued, have the
potential to improve allocative efficiency and equity across the system, particularly with the
launch of evidence-based health insurance products. In Armenia, the lack of regulation guid-
ing the systematic revision of the HBP including clarity on what factors should be considered,
stakeholders to be involved, and mechanisms for transparency, have so far hampered HBP re-
design in line with UHC goals [42]. In all three countries, HIPtool applications nurtured a pro-
cess infrastructure for future discussions on the prioritization of health resources, with a focus
on costs and coverage across the entirety of the health system. Limitations For example,
effects such as gains in productivity or school attendance cannot be captured. This means that
the positive impact of an HBP, as estimated by HIPtool, is likely to be an underestimate of the
full cross-sectoral impact. Second, since there is a trade-off between usability and flexibility,
there are many highly complex interactions between diseases and health interventions that
cannot be captured in full, as these would require more data than are available in most country
contexts. As a consequence, overlaps and synergies between the interventions included in HIP-
tool are not considered, and must be accounted for in the data being uploaded or parameteri-
sation of intervention MPIs. Also, only the static first-order impacts are currently there are many highly complex interactions between diseases and health interventions that
cannot be captured in full, as these would require more data than are available in most country
contexts. As a consequence, overlaps and synergies between the interventions included in HIP-
tool are not considered, and must be accounted for in the data being uploaded or parameteri-
sation of intervention MPIs. Also, only the static first-order impacts are currently
incorporated into the analyses. Third, HIPtool is not a disease modelling tool and therefore
does not currently account for disease progression and infectiousness, which would introduce
substantial complexity and data needs. Instead, it builds on the best existing projections of dis-
ease burden and studies of intervention effects in terms of mortality and DALYs averted. Fourth, the tool does not provide a timeline for intervention scale-up, which is instead
explored by running multiple scenarios. As such the analyses did not include a dynamic tem-
poral dimension or adjustments in effectiveness or cost at scale, but rather emphasize the pos-
sibility of reallocating resources at any given point. Fifth, as with any mathematical model,
there are numerous data limitations. For instance, the demographic data might not reflect true
population sizes in a country due to migration which is for instance significant among men in
Armenia. The analyses performed by HIPtool are only as valid as the data entered into it. How-
ever, HIPtool revealed important data gaps and “made a case” for increased focus and invest- incorporated into the analyses. Third, HIPtool is not a disease modelling tool and therefore
does not currently account for disease progression and infectiousness, which would introduce
substantial complexity and data needs. PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Limitations Instead, it builds on the best existing projections of dis-
ease burden and studies of intervention effects in terms of mortality and DALYs averted. F
th th t
l d
t p
id
ti
li
f
i t
ti
l
p
hi h i i
t
d explored by running multiple scenarios. As such the analyses did not include a dynamic tem-
poral dimension or adjustments in effectiveness or cost at scale, but rather emphasize the pos-
sibility of reallocating resources at any given point. Fifth, as with any mathematical model, there are numerous data limitations. For instance, the demographic data might not reflect true
population sizes in a country due to migration which is for instance significant among men in
Armenia. The analyses performed by HIPtool are only as valid as the data entered into it. How-
ever, HIPtool revealed important data gaps and “made a case” for increased focus and invest-
ment in the collection of data supporting decision-making. Moving forward, the UHC compendium [43] recently released by WHO provides a comprehensive database of interven-
tions and associated data that can be used as inputs in HIPtool and address a number of exist-
ing data challenges. 16 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 PLOS ONE Allocative efficiency analysis to inform health benefits package In all three model applications, the largest uncertainties were related to the ICERs. The lat-
ter are based on a large global effort but may not reflect the cost-effectiveness of the locally
delivered intervention, and the availability of ICERs as point estimates rather than ranges does
not enable uncertainty analyses. Again, HIPtool analyses can point to key ICER values which
should be refined through local studies, however, in environments where localized evidence
and analytics are limited, it may be challenging to develop valid local parameters on a scale to
inform all interventions in the HBP. This is a particularly important component in the current
optimization algorithm where the ICER value provides the basis for the estimation of effect
size and efficiency. Future iterations of the HIPtool algorithms can disassociate the effect and
efficiency parameters, but this is dependent on disaggregated cost and effects data being made
available for EUHC interventions [44]. However, the release of the UHC compendium is also
likely to enable improved future iterations of the HIPtool optimisation algorithm. Limitations Future iterations of the HIPtool algorithms can disassociate the effect and
efficiency parameters, but this is dependent on disaggregated cost and effects data being made
available for EUHC interventions [44]. However, the release of the UHC compendium is also
likely to enable improved future iterations of the HIPtool optimisation algorithm. Finally, Limitations Finally,
HIPtool is not a costing or budgeting tool, and is intended only to contribute one component
in the overall process of determining an effective HBP. Political, logistical, and other consider-
ations need to be considered outside HIPtool to determine what HBPs are feasible. Any HBP
will need to be carefully costed and the implications for implementation fully considered. These considerations are outside the scope of HIPtool, but are critical for the successful adop-
tion of an HBP. The above limitations continue to inform methodological improvements in
the HIPtool. Particular areas for methodological improvement in the tool include integrating a
more consistent intervention taxonomy, automation of calculations for current population in
need and current coverage (while maintaining functionality to manually adjust estimates) and
mechanisms for importing a greater range of estimates for intervention costs effectiveness esti-
mates. The HIPtool limitations also highlight improvements that countries can take to gener-
ate data that usefully inform priority setting. Such data systems need to collect data not only
on inputs (such as staffing and commodities) and outputs (such as number of outpatient
appointments), but integrate with epidemiological and population data to generate an under-
standing of how interventions are addressing existing demand. In addition, localized eco-
nomic evaluation and costing of a greater number of interventions will considerably improve
the applicability of results and the priority setting process in general. Nevertheless, while the
methodology is being further improved, the current HIPtool outputs can inform high-level
discussions on allocative efficiency and provide an entry point into more specific and compre-
hensive analyses in collaboration with other existing tools. In all three model applications, the largest uncertainties were related to the ICERs. The lat-
ter are based on a large global effort but may not reflect the cost-effectiveness of the locally
delivered intervention, and the availability of ICERs as point estimates rather than ranges does
not enable uncertainty analyses. Again, HIPtool analyses can point to key ICER values which
should be refined through local studies, however, in environments where localized evidence
and analytics are limited, it may be challenging to develop valid local parameters on a scale to
inform all interventions in the HBP. This is a particularly important component in the current
optimization algorithm where the ICER value provides the basis for the estimation of effect
size and efficiency. PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 Acknowledgments We thank the stakeholders in Armenia, Cote d’Ivoire and Zimbabwe for their participation in
the analyses at all stages from design to completion, and the peer reviewers of the three country
studies for providing comments that helped improve the quality of this manuscript. We also
thank Lara Gosce´, Shepherd Shamu, Chenjerai N. Sisimayi, Laurence Lannes, Cliff Kerr, Has-
san Haghparast-Bidgoli, Marianna Koshkakaryan, Lusine Yengibaryan for their invaluable
contributions to the respective country studies. The findings, interpretations, and conclusions
expressed in this paper are entirely those of the authors. They do not necessarily represent the
views of the World Bank and its affiliated organizations, or those of the Executive Directors of
the World Bank or the governments they represent. Conclusions There is a clear need to set health sector priorities, especially now that COVID-19 has further
constrained fiscal space and increased the burden on health systems globally. A recent global
health investment analysis suggested that where governments maintain pre-COVID 19 trends
in health spending, the share of government resources flowing to health will have to increase
on average by more than 11% above pre-COVID levels [45]. A growing number of countries
are defining HBPs to allocate limited resources, illustrated here by the case of Cote d’Ivoire, or
review their HBPs (Armenia and Zimbabwe cases). A HIPtool application within a country,
serves to demonstrate the gains in terms of health impact and related health system expendi-
ture, that can be achieved by taking an allocative efficiency conceptual approach to HBP
design. While allocative efficiency tools such as HIPtool cannot replicate complex system reali-
ties due to data and tool limitations, they allow testing of policy options and can provide useful
directional results. Equally important, they can give structure to the health policy process,
mobilize stakeholders and centralize an array of health data, if well implemented. The HIPtool
findings may also have implications for donor coordination and effective allocation of donor PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 17 / 21 17 / 21 PLOS ONE Allocative efficiency analysis to inform health benefits package funding. Decision support tools like HIPtool, combined with other technical analyses and con-
sideration for political and implementation realities, will provide the necessary evidence to
improve health resource allocations, transition from passive to strategic purchasing, and
achieve more health for health spending. Supporting information pp
g
S1 Appendix. HIPtool technical specifications. (PDF)
S2 Appendix. HIPtool interventions by delivery platform and DCP3 care package, with
respective global burden of disease causes addressed. (PDF)
S3 Appendix. Local equity and financial risk protection scores (Armenia). (PDF)
S4 Appendix. Additional optimization results. (PDF) S3 Appendix. Local equity and financial risk protection scores (Armenia).
(PDF) S3 Appendix. Local equity and financial risk protection scores (Armenia). (PDF) S4 Appendix. Additional optimization results. (PDF) S2 Appendix. HIPtool interventions by delivery platform and DCP3 care package, with
respective global burden of disease causes addressed.
(PDF) S3 Appendix. Local equity and financial risk protection scores (Armenia). (PDF) S1 Appendix. HIPtool technical specifications.
(PDF) S2 Appendix. HIPtool interventions by delivery platform and DCP3 care package, with
respective global burden of disease causes addressed. (PDF) References 1. The World Bank. Universal Health Coverage Study Series (UNICO), 2017. Accessed 15 May 2017. http://www.worldbank.org/en/topic/health/publication/ universal-health-coverage-study-series 2. Glassman A, Chalkidou K. Priority-setting in health: building institutions for smarter public spending. Washington D.C 2012. Accessed 1 Dec 2015. http://www.cgdev.org/publication/priority-setting-health-
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Gerard J. Abou Jaoude, Adanna Chukwuma. Methodology: Gerard J. Abou Jaoude, David P. Wilson. Methodology: Gerard J. Abou Jaoude, David P. Wilson. Methodology: Gerard J. Abou Jaoude, David P. Wilson. Project administration: Xiaohui Hou, Adanna Chukwuma, Christine Lao Pena, Opope O. Tshivuila Matala. Software: Xiaohui Hou, Gerard J. Abou Jaoude, Jolene Skordis. Software: Xiaohui Hou, Gerard J. Abou Jaoude, Jolene Skordis. Supervision: Xiaohui Hou, Jolene Skordis. Supervision: Xiaohui Hou, Jolene Skordis. Validation: Thomas Wilkinson, Denizhan Duran. Visualization: Nicole Fraser-Hurt, Thomas Wilkinson. Visualization: Nicole Fraser-Hurt, Thomas Wilkinson. Writing – original draft: Nicole Fraser-Hurt, Xiaohui Hou. Writing – original draft: Nicole Fraser-Hurt, Xiaohui Hou. 18 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
November 29, 2021 PLOS ONE | https://doi.org/10.1371/journal.pone.0260247
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Duran, Gerard J. Abou Jaoude, Jolene Skordis, Adanna Chukwuma, Christine Lao Pena,
Opope O. Tshivuila Matala, Marelize Gorgens, David P. Wilson. Writing – review & editing: Nicole Fraser-Hurt, Xiaohui Hou, Thomas Wilkinson, Denizhan
Duran, Gerard J. Abou Jaoude, Jolene Skordis, Adanna Chukwuma, Christine Lao Pena,
Opope O. Tshivuila Matala, Marelize Gorgens, David P. Wilson. References Accessed 2 March 2021, http://dcp-3.org/resources/costs-
and-affordability-essential-universal-health-coverage-low-and-middle-income 14. Watkins DA, Norheim OF, Jha P, Jamison DT. Reducing Mortality within Universal Health Coverage:
The DCP3 Model. DCP3 Working Paper Series, 2017. Working Paper #21. Accessed 22 April 2021,
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middle-income 15. Center for the Evaluation of Value and Risk in Health, Tufts Registry: https://cevr.tuftsmedicalcenter. org/databases/gh-cea-registry 16. WHO UHC compendium here as it is a more comprehensive alternative and data can equally be input-
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Armenia, 2020. World Bank, Washington, DC. Retrieved February 2, 2021 https://openknowledge. worldbank.org/handle/10986/34491 43. UHC Compendium: Health interventions for Universal Health Coverage. Accessed 14 March 2021. https://www.who.int/universal-health-coverage/compendium 44. Arnold M, Griffin S, Ochalek J, Revill P, Walker S. A one stop shop for cost-effectiveness evidence? Recommendations for improving Disease Control Priorities. Cost Effectiveness and Resource Alloca-
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Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria
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Egyptian Academic Journal of Biological Sciences. G, Microbiology
| 2,015
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cc-by
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Citation: Egypt. Acad. J. Biolog. Sci. (G. Microbiolog) Vol.7 (1)pp. 101-109(2015) ABSTRACT ARTICLE INFO
Article History
Received: 1/11/2015
Accepted: 10/12/2015
_________________
Keywords:
Honey ,
Antibiotics ,
Gram Positive Bacteria,
Synergistic Effects. Antibacterial Activity This study aimed to investigate the growth inhibitory effect of
sidr and sommor honeys and to evaluate the synergistic effects of
various honey concentrations and five antibiotics on gram-positive
bacteria in vitro using agar well diffusion and disk diffusion
techniques. This is the first time for studying the antibacterial activity
and synergistic effect of sommor honey against bacteria. The results
indicated that sidr and sommor honeys had antibacterial activity
against the clinical isolates and reference strains of S. aureus and
Strept. pyogenes. The reference strains were more susceptible to sidr
honey than sommor honey while the clinical isolates were more
susceptible to sommor honey than sidr honey. Increasing honey
concentrations either alone or in combination with antibiotics was
significantly increased (P<0.05) the growth inhibition of the tested
bacteria. The synergistic effects of honeys with antibiotics were
significantly (P<0.05) different among the tested gram-positive
bacteria. The highest synergistic effect was observed against Strept. pyogenes clinical isolate when sidr and sommor honeys combined with
ofloxacin,
pipracillin,
amoxicillin
+
clavulanic
acid
and
sulphamethoxazole + trimethoprim. It can be concluded that sidr and
sommor honeys improved the antibiotic activity and presented a new
avenue for treatment of gram-positive bacterial infections. yptian Academic Journal of Biological Sciences is the official English language journal of
the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of
Sciences Ain Shams University.
icrobiology journal is one of the series issued twice by the Egyptian Academic Journal of
iological Sciences, and is devoted to publication of original papers related to the research
ross the whole spectrum of the subject. These including bacteriology, virology, mycology
d parasitology. In addition, the journal promotes research on the impact of living organisms
on their environment with emphasis on subjects such a resource, depletion, pollution,
biodiversity, ecosystem…..etc
www.eajbs.eg.net
Provided for non-commercial research and education use.
Not for reproduction, distribution or commercial use.
Vol. 7 No. 1 (2015) Eg ptian Academic Jo rnal of Biological Sciences is the official English lang age jo rnal of
Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use. Vol. 7 No. 1 (2015) Vol. 7 No. 1 (2015) Egyptian Academic Journal of Biological Sciences is the official English language journal of
the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of
Sciences Ain Shams University. Microbiology journal is one of the series issued twice by the Egyptian Academic Journal of
Biological Sciences, and is devoted to publication of original papers related to the research
across the whole spectrum of the subject. These including bacteriology, virology, mycology
and parasitology. In addition, the journal promotes research on the impact of living organisms
on their environment with emphasis on subjects such a resource, depletion, pollution,
biodiversity, ecosystem…..etc www.eajbs.eg.net Citation: Egypt. Acad. J. Biolog. Sci. (G. Microbiolog) Vol.7 (1)pp. 101-109(2015) Egypt. Acad. J. Biolog. Sci., 7(1): 101 – 109 (2015)
Egyptian Academic Journal of Biological Sciences
G. Microbiology
ISSN: 2090-0872
www.eajbs.eg.net
Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria Egypt. Acad. J. Biolog. Sci., 7(1): 101 – 109 (2015) Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positiv Masoud, E. A.* Alqurashi, A. M. and Alamin, A. A
Department of Applied Medical Sciences, Community College, Najran University, Saudi Arabia. *Corresponding author E-mail: husseinea1968@yahoo.com Citation: Egypt. Acad. J. Biolog. Sci. (G. Microbiolog) Vol.7 (1)pp. 101-109(2015) INTRODUCTION Infectious diseases continue to take a toll on human health and life expectancy. Treatment of these infections is becoming increasingly difficult due to antibiotic resistance to
currently available drugs (Saleem et al., 2010). The emergence in recent years of numerous
resistant strains of pathogenic bacteria to a range of formerly efficient antibiotics constitutes a
serious threat to public health (Raymond et al., 2011). Honey originally used by the ancient
Egyptians and Greeks, honey is a viscous, saturated sugar solution now widely used in wound
care (Simon et al., 2009). Bees and honey are mentioned in the holy Qur'an and there is even
one Chapter in the Qur'an named "Surah an-Nahl" meaning bees. Allah Said (what means):
"And the Lord inspired the bee, saying: Take your habitations in the mountains and in the
trees and in what they erect. Then, eat of all fruits and follow the ways of your Lord made
easy (for you)'. There comes forth from their bellies a drink of varying colors wherein is
healing for men. Citation: Egypt. Acad. J. Biolog. Sci. (G. Microbiolog) Vol.7 (1)pp. 101-109(2015) Masoud, E. A. et al. 102 Verily in this is indeed a sign for
people who think." (Holy Qur'an 16:68-69). The mechanisms of action of honey are still
not
fully
understood;
however,
the
antimicrobial activity of most honeys is
linked to the production of hydrogen
peroxide by the enzyme glucose oxidase
which combined with high acidity and exerts
an antimicrobial effect (French et al., 2005). Honey is an ancient wound treatment that
was re-introduced into modern medical
practice and possesses therapeutic potential
and antimicrobial activity (Andargarchew et
al., 2004). There are many reports of
bactericidal and bacteriostatic activity of
honey
against
bacteria,
which
have
developed resistance to many antibiotics
(Patton et al., 2006). Sidr honey is made
from bees who feed only on the nectar of the
sidr tree, which is native in the South Saudi
Arabia and Yemen regions. Sidr honey has
wide medicinal applications and uses which
include: liver diseases, stomach ulcers,
respiratory infections, malnutrition diseases,
digestive
problems,
constipation,
eye
diseases, infected wounds and burns. Sidr
honey
has
strong
antioxidant
and
antibacterial properties (Alandejani et al.,
2009). By searching the internet, no
published papers were recorded for sommor
honey, and just short data was reported at the
web sites of honey markets. MATERIALS AND METHODS Preparation of honey concentrations,
Confirmation
of
isolates
identification,
antibacterial
assays,
antimicrobial
susceptibility pattern and evaluation of the
synergistic
effect
of
various
honey
concentrations with antibiotics on gram
positive bacteria were conducted at the
Microbiology laboratory, Department of
Applied
Medical
Sciences,
Community
College, Najran University during February
to August 2015. INTRODUCTION Sommor honey
is made from bees who feed on sommor tree
(Acacia) in the Hadramout region, Yemen. A
large number of honeys are available in the
Saudi market and some of them are
traditionally used as remedy for several
ailments. To date, the antibacterial efficiency
of local Saudi honeys has not been
thoroughly evaluated (Eman and Mohamed,
2011). To combat antibiotic resistance,
combination antibiotic treatment is widely
practiced in the clinic. Such treatment can
result in synergism to provide increased
efficacy and a reduction in amount of each
antibiotic used, which can reduce the risk of
possible side effects and treatment costs (Lee
et al., 2007; Wagner and Merzenich, 2009;
Leibovici et al., 2010). A combination of the antimicrobial
properties
of
clinically
approved antibiotics and the antibacterial
activity of honey could lead to a new
spectrum of antimicrobials that have the
potential to prevent the emergence of
resistant bacterial strains, providing broad-
spectrum
coverage
and
consequently
improving therapeutic efficiency (Patrick et
al.., 2013). Moreover, combination use of
antibiotics with different modes of action
reduce the risk of antibiotic resistance arising
during therapy (Raha, 2006). In medical
practice, honey might be delivered in
combination with traditional antibiotics, with
both being administered topically or with
honey being administered topically and the
traditional antibiotic(s) being administered
systemically. Therefore, it is useful to
understand the interaction between honey
and conventional antibiotics (Michelle and
Robin, 2014). Our goals were to assay the
growth inhibitory effect of sidr and sommor
honeys and to study the synergistic effects of
various honey concentrations and antibiotics
on gram-positive bacteria in vitro. Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria 103 This was done by dissolving the respective
volumes: 1, 2, 4, 6, 8 mL of each honey
sample into corresponding volumes of sterile
distilled water to give finally a 10 mL
preparation. room temperature; the medium was punched
with
six
millimeters
diameter
wells. Different concentrations (10, 20, 40, 60 and
80%) of sidr and sommor honeys were
checked for growth inhibitory effect by
introducing 50 µl into the well and allowed
to diffuse at room temperature for 20
minutes. The
plates
were
incubated
aerobically at 37° C for 24 hrs. and the
inhibition
zone
diameter
(IZD)
was
measured (mm) using a ruler. Tests were
done in triplicate and the growth inhibitory
effect of different honey samples and
concentrations were recorded. Amikacin
(Oxoid) antibiotic disc (30 µg) was used as a
positive control. Bacterial isolates and culture media Four-gram positive bacteria were used
in this study. Two clinical pathogenic
bacterial isolates, S. aureus and Strept. Pyogenes
were
obtained
from
the
Microbiology
laboratory,
King
Khalid
Hospital (K.K.H), Najran region, Saudi
Arabia. The organisms were identified by an
automated system (Micro Scan Walkaway,
Siemens) and the results were confirmed
(Koneman et al., 1992). Two reference
strains, S. aureus ATCC14775 and Strept. pyogenes ATCC19615 were obtained from
Microbiologics®. The
isolates
were
subcultured on appropriate agar plates 24
hrs. before antibacterial tests. Brain Heart
Infusion broth (Oxoid, England), Mueller
Hinton agar (Oxoid, England) and Nutrient
agar (Oxoid, England) were used in this
investigation. Media
were
prepared
according to manufacture recommendations. Antibacterial activity of honey samples Honey
samples
and
preparation
of
concentrations Sidr
and
sommor
honeys
were
purchased from honey markets at Najran
city, Saudi Arabia. Najran is a region of
Saudi Arabia, located in the south of the
country along the border with Yemen. It has
an area of 119,000 km². Its capital is Najran. Various concentrations of each honey
constituting, 10, 20, 40, 60 and 80% (v/v)
were prepared using sterile distilled water. Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria RESULTS AND DISCUSSION Medicinal honeys are registered for
topical application in several countries and
are formulated in tubes into ointments and
gels, and made into wound dressings
(Kwakman et al., 2011). Table 2: Growth inhibitory effect of various sidr honey concentrations and amikacin on gram-positive bacteria
(all values in mm). Bacteria
Sidr honey concentrations
Amikacin
80%
60%
40%
20%
10%
S. aureus ATCC 14775
15.33 ± 0.33 a
13.33 ± 0.33 b
11.00 ± 0.00 c
00.00 ± 0.00
00.00 ± 0.00
21.00 ± 0.00
Strept. pyogenes ATCC 19615
16.00 ± 0.58 d
14.33 ± 0.33 e
12.33 ± 0.33 f
00.00 ± 0.00
00.00 ± 0.00
21.33 ± 0.33
S. aureus
16.67 ± 0.33 d
14.67 ± 0.33 e
13.00 ± 0.00 b
00.00 ± 0.00
00.00 ± 0.00
22.67 ± 0.33
Strept. pyogenes
18.33 ± 0.33 g
16.00 ± 0.00 d
13.33 ± 0.33b
12.00 ± 0.00 d
10.33 ± 0.33 c
23.67 ± 0.33
a-g Values with different superscripts in the same raw and column differ at p< 0.05. hibitory effect of various sidr honey concentrations and amikacin on gram-positive bacteria
) Table 3: Growth inhibitory effect of various sommor honey concentrations and amikacin on gram-positive
bacteria (all values in mm). Bacteria
Sommor honey concentrations
Amikacin
80%
60%
40%
20%
10%
S. aureus ATCC 14775
12.67 ± 0.33 a
10.33 ± 0.33 b
8.67 ± 0.33 c
00.00 ± 0.00
00.00 ± 0.00
21.00 ± 0.00
Strept. pyogenes ATCC 19615
11.33 ± 0.33 d
11.00 ± 0.58 d
8.67 ± 0.33 c
00.00 ± 0.00
00.00 ± 0.00
21.33 ± 0.33
S. aureus
18.00 ± 0.00 e
16.00 ± 0.00 f
14.33 ± 0.33 g
12.67 ± 0.33 a
11.00 ± 0.00 d
22.67 ± 0.33
Strept. pyogenes
35.33 ± 0.33 h
31.00 ± 0.58 i
18.00 ± 0.33 g
15.67 ± 0.33 f
13.33 ± 0.33 c
23.67 ± 0.33
a-i Values with different superscripts in the same raw and column differ at p< 0.05. Growth inhibitory effect of various sommor honey concentrations and amikacin on gram-positive
acteria (all values in mm) Table 3: Growth inhibitory effect of various sommor honey concentrations and amikacin o
bacteria (all values in mm). Chapagain, (2014) found that the clinical
isolate was more sensitive to manuka honey
than the reference strain. Statistical analysis Data were analyzed statistically and
were presented as means ± S.E. (Standard
Error) using an analysis of variance
(ANOVA) according to Fisher's PLSD test. Differences were considered significant
when p<0.05. Antibiotic susceptibility testing Antibacterial
susceptibility
pattern
was
performed
using
disk
diffusion
technique (Bauer et al.., 1966). Five
antibiotics were used in this study (Table 1). Each bacterial isolate, at a concentration of
1.5 × 106 cells/ml (adjusted to the 0.5
McFarland
turbidity
standards)
was
inoculated by streaking the swab over
surface of Mueller Hinton agar plates. The
plates were allowed to dry for 5 minutes at
room temperature. Then, all antibiotic disks
were placed on the surface of the plates and
pressed gently to ensure complete contact
with agar medium. After 15 minutes of
placing disks, the plates were incubated for
24 hrs. at 37 °C. The assessment of bacterial
susceptibility was based on measurement of
inhibition zone diameter (mm) formed
around the antibiotic disk. The experiment
was
repeated
in
triplicates
for
each
microorganism. The growth inhibitory effect of sidr
and sommor honeys was determined by the
agar well diffusion technique according to
NCCLS (1993). Clinical and reference
bacterial cultures were grown in Brain Heart
Infusion broth at 37 °C for 4 hrs. Each
isolate, at a concentration of 1.5 × 106
cells/ml (adjusted to the 0.5 McFarland
turbidity standards) was inoculated onto the
surface of Mueller Hinton agar plates using
swabs. The plates were allowed to dry at Table 1: List of antibiotics
Antibiotic
Symbol
Potency( µg)
Company
Ofloxacin
OFX
5
Oxoid
Imipenem
IPM
10
Oxoid
Pipracillin
PRL
100
Oxoid
Amoxicillin + Clavulanic acid (2:1)
AMC
30
Oxoid
Sulphamethoxazole (23.75 µg)+ Trimethoprim(1.25 µg)
SXT
25
Oxoid Evaluation the synergistic effect of various
honey concentrations and antibiotics Clinical isolates and reference strains
at a concentration of 1.5 × 106 cells/ml
(adjusted to the 0.5 McFarland turbidity Masoud, E. A. et al. 104 Antibacterial activity of sidr and sommor
honeys Antibacterial activity of sidr and sommor
honeys standards) were inoculated onto the surface
of Mueller Hinton agar plates using swabs. Thereafter, the antibiotic disks of 6 mm in
diameter were placed on the surface of each
inoculated plate then saturated with 10 µl of
various honey concentrations (40, 60 and
80%). The plates were incubated for 24 hrs. at 37°C. Inhibition zone diameters were
recorded and compared with that of the
antibiotic alone (Betoni et al., 2006). Statistical analysis y
The
growth
inhibitory
effect
of
different
sidr
and
sommor
honey
concentrations was presented in Tables (2-3). All the tested gram-positive bacteria were
susceptible to sidr and sommor honeys at
concentrations 80-40%. Basualdo et al.,
(2007) reported that the bactericidal effect of
honey is to be dependent on concentration of
honey used and the nature of the bacteria. Khan et al., (2007) mentioned that the
antibacterial property of honey is also
derived from the osmotic effect of its high
sugar content and low moisture content,
along with its acidic properties of gluconic
acid and the antiseptic properties of its
H2O2.Various sidr honey concentrations (80-
10%) inhibited the growth of Strept. pyogenes
clinical
isolate
despite
that
different sommor honey concentrations (80-
10%) exhibited antimicrobial effects against
S. aureus and Strept. pyogenes clinical
isolates. Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics against S. aureusATCC14775
reference strain Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics against S. aureusATCC14775
reference strain All the tested antibiotics inhibited the
growth of S. aureus ATCC14775 with IZDs Table 4: Synergistic effect of sidr and sommor honeys (80 - 40%) with antibiotics on S. aureus A
l
i
) c effect of sidr and sommor honeys (80 - 40%) with antibiotics on S. aureus ATCC14775 (all
) Table 4: Synergistic effect of sidr and sommor honeys (80 - 40%) with antibiotics on S. aureus ATCC14775 (all
values in mm). Antibiotics+ Honeys
Honey concentrations
Antibiotic alone
80%
60%
40%
OFX + Sidr
OFX + Sommor
29.33 ± 0.33
29.33± 0.33
29.00 ± 0.00
29.33 ± 0.33
29.33± 0.33 a
28.33± 0.33 b
28.33 ± 0.33 b
29.33 ± 0.33 a
IPM + Sidr
IPM + Sommor
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
PRL + Sidr
PRL + Sommor
26.67 ± 0.33 c
25.33 ± 0.33 d
25.00 ± 0.00 d
20.33± 0.33 e
24.00 ± 0.00 f
22.33 ± 0.33 e
22.33 ± 0.33 e
20.33± 0.33 e
AMC + Sidr
AMC + Sommor
33.67± 0.33 g
32.00 ± 0.00 h
30.67± 0.33 h
26.67 ± 0.33 c
31.00 ± 0.00 h
29.33 ± 0.33 a
29.00 ± 0.58 a
26.67 ± 0.33 c
SXT + Sidr
SXT + Sommor
31.67 ± 0.33 h
30.00 ± 0.00 a
29.00 ± 0.58 a
25.33 ± 0.33 d
28.00 ± 0.00 b
28.00 ± 0.00 b
27.67 ± 0.58 b
25.33 ± 0.33 d
a-h Values with different superscripts in the same raw and column differ at p< 0.05. a-h Values with different superscripts in the same raw and column differ at p< 0.05. Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics
against
Strept. pyogenes
ATCC19615 reference strain RESULTS AND DISCUSSION Taormina et al.,
(2001) reported that physical properties
along with geographical distribution and
different floral sources may play important
role in the antimicrobial activity of honey. Increasing honey concentrations significantly Our
findings
agree
with
other
observations (Wilkinson and Cavanagh,
2005; Mohammad et al., 2008; Henriques et
al., 2010; Maddocks et al., 2012). The
reference strains were more susceptible to
sidr honey than sommor honey. On the other
hand, the clinical isolates more susceptible to
sommor honey than sidr honey. Jenkins and Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria 105 ranged from 20.33± 0.33-52.00±0.00 mm
(Table 4). It was observed that various sidr
and sommor honey concentrations (80-40%)
exerted synergistic effects with pipracillin,
amoxicillin
+
clavulanic
acid
and
sulphamethoxazole + trimethoprim. The
present findings revealed that sidr and
sommor
samples
at
((80-40%)
concentrations did not show synergistic
effects with ofloxacin and imipenem on S. aureus ATCC14775. Our results agree with
other observations (Ali et al., 2005; Patrick
et al., 2013). increased the growth inhibition of the tested
bacteria. This result agrees with the result
previously recorded by (Alqurashi et al.,
2013). The clinical and reference strains
were susceptible to amikacin antibiotic with
IZDs ranged from 21.00±0.00 - 23.67 ± 0.33
mm. a-g Values with different superscripts in the same raw and column differ at p< 0.05. Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics
against
Strept.
pyogenes
ATCC19615 reference strain Synergistic action was observed
when different sidr and sommor honey
concentrations (80-40%) were combined
with
ofloxacin,
imipenem
and
sulphamethoxazole + trimethoprim on S. aureus clinical isolate. No synergism was observed for
combination
of
honey
samples
with
imipenem and amoxicillin + clavulanic acid. Sommor honey had synergistic effect on
Strept. pyogenes ATCC19615 reference
strain when combined with ofloxacin. In
contrast, no synergistic effect resulting from
combination of sidr honey with ofloxacin. Jenkins and Chapagain, (2014) found that
Combining each of the antibiotics with
manuka honey resulted in either synergistic
or additive activity for a reference strain of
Strept. pyogenes (NCTC 10085) reference
strain and a clinical isolate of Strept. pyogenes (74721). Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics against S. aureus clinical isolate g
The results of the growth inhibitory
effect of antibiotics and their synergism with
different
sidr
and
sommor
honey
concentrations on S. aureus clinical isolate
were presented in Table 6. The present
findings revealed that S. aureus clinical
isolate was sensitive to the five tested
antibiotics. Synergistic action was observed
when different sidr and sommor honey
concentrations (80-40%) were combined
with
ofloxacin,
imipenem
and
sulphamethoxazole + trimethoprim on S. aureus clinical isolate. Table 6: Synergistic effect of sidr and sommor honeys (80 - 40%) with antibiotics on S. aureus (all values in
mm). Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics
against
Strept.
pyogenes
ATCC19615 reference strain Antibiotics+ Honeys
Honey concentrations
Antibiotic alone
80%
60%
40%
OFX + Sidr
OFX + Sommor
35.67 ± 0.33 a
33.67 ± 0.33 b
32.00 ± 0.00 c
28.67 ± 0.33 d
36.33 ± 0.33 a
34.33 ± 0.33 b
32.00 ± 0.58 c
28.67 ± 0.33 d
IPM + Sidr
IPM + Sommor
42.00 ± 0.00 e
39.33 ± 0.33 f
39.67 ± 0.33 f
35.67 ± 0.33 a
41.33 ± 0.33 g
39.33 ± 0.33 f
38.67 ± 0.33 f
35.67 ± 0.33 a
PRL + Sidr
PRL + Sommor
28.33 ± 0.33
27.67 ± 0.33
27.67 ± 0.33
28.00 ± 0.00
28.00 ± 0.00
28.00 ± 0.58
27.67 ± 0.33
28.00 ± 0.00
AMC + Sidr
AMC + Sommor
32.00 ± 0.00 c
31.33 ± 0.33 c
31.33 ± 0.33 c
30.67 ± 0.33 h
31.33 ± 0.33 h
30.67 ± 0.67 h
30.67 ± 0.33 h
30.67 ± 0.33 h
SXT + Sidr
SXT + Sommor
40.00 ± 0.58 f
37.67 ± 0.33 i
37.00 ± 0.00 j
32.67 ± 0.67 c
39.67 ± 0.33 f
37.33 ± 0.33 i
36.67 ± 0.33 j
32.67 ± 0.67 c
a-jValues with different superscripts in the same raw and column differ at p< 0.05. fect of sidr and sommor honeys (80 - 40%) with antibiotics on S. aureus (all values in Combination of various honey samples
with pipracillin and amoxicillin + clavulanic
acid did not show any synergism on S. aureus clinical isolate. Synergism between
honey and antibiotics was documented in
previous studies (Patrick et al., 2013;
Michelle and Robin, 2014). showed on Strept. pyogenes clinical isolate
when various honey concentrations were
combined
with
ofloxacin,
pipracillin,
amoxicillin
+
clavulanic
acid
and
sulphamethoxazole + trimethoprim. It was observed that, no synergism was
obtained against Strept. pyogenes clinical
isolate when honey samples were combined
with imipenem. Our findings were supported
by the results previously reported by
(Jenkins and Cooper, 2012; Jenkins and
Chapagain, 2014). Our results further show
that there was an increase of IZD for the
tested microorganisms with the increase of
honey concentration either alone or in
combination with antibiotics. Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics against Strept. pyogenes clinical
isolate Synergistic effect of different sidr and
sommor honey concentrations (80 – 40%)
with antibiotics and sensitivity of Strept. pyogenes clinical isolate to antibiotics alone
were summarized in Table 7. Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics
against
Strept.
pyogenes
ATCC19615 reference strain presented in Table 5. The results
showed that Strept. pyogenes ATCC19615
reference strain was sensitive to the tested
antibiotics. Various sidr and sommor honey
concentrations (80-40%) had synergistic
effect
with
pipracillin
and
sulphamethoxazole + trimethoprim on Strept. pyogenes ATCC19615 reference strain. presented in Table 5. The results
showed that Strept. pyogenes ATCC19615
reference strain was sensitive to the tested
antibiotics. Various sidr and sommor honey
concentrations (80-40%) had synergistic
effect
with
pipracillin
and
sulphamethoxazole + trimethoprim on Strept. pyogenes ATCC19615 reference strain. The susceptibility of Strept. pyogenes
ATCC19615 reference strain to Antibiotics
and synergistic effect of sidr and sommor
honey
samples
with
antibiotics
were Table 5: Synergistic effect of sidr and sommor honeys (80 - 40%) with antibiotics on Strept. pyogene
ATCC19615 (all values in mm). Antibiotics+ Honeys
Honey concentrations
Antibiotic alone
80%
60%
40%
OFX + Sidr
OFX + Sommor
32.00 ± 0.00 a
30.67 ± 0.33 a
30.33 ± 0.33 a
31.33 ± 0.33 a
36.67 ± 0.33 b
34.33 ± 0.33 c
33.67 ± 0.33 c
31.33 ± 0.33 a
IPM + Sidr
IPM + Sommor
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
PRL + Sidr
PRL + Sommor
48.67 ± 0.33 d
46.33 ± 0.33 e
45.00 ± 0.00 f
40.33 ± 0.33 g
49.00 ± 0.00 d
46.67 ± 0.33 e
44.00 ± 0.00 f
40.33 ± 0.33 g
AMC + Sidr
AMC + Sommor
46.33 ± 0.33 e
46.33 ± 0.00 e
46.00 ± 0.00 e
46.00 ± 0.00 e
45.33 ± 0.33 f
45.00 ± 0.00 f
45.67 ± 0.00 f
46.00 ± 0.00 e
SXT + Sidr
SXT + Sommor
38.67 ± 0.33 g
36.00± 0.00 b
34.00 ± 0.58 c
31.67 ± 0.33 a
36.33 ± 0.33 b
34.33 ± 0.33 c
34.67 ± 0.67 c
31.67 ± 0.33 a
a-g V l
i h diff
i
i
h
d
l
diff
0 05 Masoud, E. A. et al. 106 Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics against S. aureus clinical isolate
The results of the growth inhibitory
effect of antibiotics and their synergism with
different
sidr
and
sommor
honey
concentrations on S. aureus clinical isolate
were presented in Table 6. The present
findings revealed that S. aureus clinical
isolate was sensitive to the five tested
antibiotics. Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria Synergistic Effects of Honeys and Commonly Used Antibiotics on Gram Positive Bacteria 107 Table 7: Synergistic effect of sidr and sommor honeys (80 - 40%) with antibiotics on Strept. Pyogenes (all
values in mm). Antibiotics+ Honeys
Honey concentrations
Antibiotic alone
80%
60%
40%
OFX + Sidr
OFX + Sommor
37.33 ± 0.33 a
36.33 ± 0.33 b
34.00 ± 0.00 c
28.00 ± 0.00 d
36.00 ± 0.00 b
34.33 ± 0.33 c
32.33 ± 0.33 e
28.00 ± 0.00 d
IPM + Sidr
IPM + Sommor
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
52.00 ± 0.00
PRL + Sidr
PRL + Sommor
52.33 ± 0.33 f
50.33 ± 0.33 g
47.67 ± 0.33 h
42.00 ± 0.00 i
50.67 ± 0.33 j
46.00 ± 0.58 k
45.00 ± 0.00 l
42.00 ± 0.00 i
AMC + Sidr
AMC + Sommor
56.00 ± 0.00 m
55.00 ± 0.00 n
51.67 ± 0.33 f
43.67 ± 0.33 o
55.67 ± 0.33 g
54.00 ± 0.00 f
49.33 ± 0.33 e
43.67 ± 0.33 o
SXT + Sidr
SXT + Sommor
36.33 ± 0.33 b
34.33 ± 0.33 c
32.00 ± 0.33 e
29.33 ± 0.33 p
36.67 ± 0.89 b
35.00 ± 0.58 c
32.00 ± 0.00 e
29.33 ± 0.33 p
a-p Values with different superscripts in the same raw and column differ at p< 0.05. REFERENCES Alandejani, T., Marsan, J., Ferris, W., Slinger, R. and Chan, F. (2009). Effectiveness of honey
on Staphylococcus aureus and Pseudomonas
aeruginosa biofilms. Otolaryngol Head
Neck Surg, 141(1):114-8 Alandejani, T., Marsan, J., Ferris, W., Slinger, R. and Chan, F. (2009). Effectiveness of honey
on Staphylococcus aureus and Pseudomonas
aeruginosa biofilms. Otolaryngol Head
Neck Surg, 141(1):114-8 Ali, A. Al-Jabri., Sumaya, A. Al-Hosni., Basil,
C. Nzeako., Zahra, H. Al-Mahrooqi. and
Herbert
Nsanze. (2005). Antibacterial
activity of Omani honey alone and in
combination with gentamicin. Saudi Med J,
26(5):767-771 p
y
Funding and policy This study was sponsored and funded
by Deanship of Scientific Research, Najran
University, Saudi Arabia (Grant Code:
NU/ESCI/14/014). Authors' Contribution All authors participated in designing
this study. Elsayed Masoud collected honey
samples, drafted and revised the manuscript. Elsayed Masoud and Mohammadeen Alamin
performed the experiments. Mohammadeen
Alamin conducted the statistical analysis. Abdulrahman
Alqurashi
collected
the
clinical isolates. The authors shared in the
interpretation of study. The present study concluded that sidr
and sommor honeys at concentrations (80-
40%) had antibacterial activity against
clinical and reference strains of S. aureus
and Strept. pyogenes. The clinical isolates
were more sensitive to sidr and sommor
honeys than references strains. Both honey
samples in various concentrations (80-10%)
inhibited the growth of Strept. pyogenes
clinical isolate than the other tested bacteria. The growth inhibition of the tested bacteria
was significantly increased (P<0.05) by
increasing honey concentrations either alone
or in combination with antibiotics. The
synergistic effects of sidr and sommor
honeys combined with antibiotics were
differed among the investigated organisms. The highest synergistic effect was observed
against Strept. pyogenes clinical isolate when
sidr and sommor honeys combined with
ofloxacin,
pipracillin,
amoxicillin
+
clavulanic acid and sulphamethoxazole +
trimethoprim. Sidr and sommor honeys were
effective antibacterial agents and can be used
in combination with antibiotics for treatment
of
gram
positive
bacterial
infections
especially S. aureus and Strept. pyogenes. ACKNOWLEDGMENTS The authors thank Najran University,
Saudi Arabia for its financial support of this
study. Special thank for Prof. Adil Al –
Garrai for advice in statistical analysis. Antibiotic susceptibility and synergistic
effect of various honey concentrations and
antibiotics
against
Strept.
pyogenes
ATCC19615 reference strain The present
study revealed that the synergism was Competing interest The authors declare that there is no
conflict of interests regarding the publication
of this paper. Alqurashi, A. M., Masoud, E. A. and Alamin, M. A. (2013). Antibacterial activity of Saudi Alqurashi, A. M., Masoud, E. A. and Alamin, M. A. (2013). Antibacterial activity of Saudi 108 Masoud, E. A. et al. (1992). Packaged in Kit Identification
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Philadelphia, PA., 163-170 (1992). Packaged in Kit Identification
System. Color Atlas and Textbook of
Diagnostic Microbiology. Koneman, E.W. (Eds.),
4th
Edn. B. Lippincott
Co.,
Philadelphia, PA., 163-170 honey
against
Gram-negative
bacteria. Journal
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and
Antimicrobials,
5(1):1-5
doi:10.5897/JMA2012.0235 Andargarchew, M., Belay, T. and Fetene, D. (2004). In
vitro
assessment
of
the
antimicrobial potential of honey on common
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18:107 - 11 Kwakman, P.H.S., te Velde, A. A., de Boer, L.,
V and enbroucke-Grauls, C.M.J.E., and
Zaat, S.A.J. (2011). Two major medicinal
honeys have different mechanisms of
bactericidal activity. PLoS One, 6(3):
ArticleIDe17709 Basualdo, C., Sgroy, V., Finola, M.S., andJuam,
M. (2007). Comparison of the antibacterial
activity of honey from different provenance
against bacteria usually isolated from skin
wounds. Vet Microbiol, 124:375-381 Lee J, J.i. Y., Lee, S. and Lee, I. (2007). Effect of
Saliva miltiorrhizabunge on antimicrobial
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45: 350 Bauer, A.W., Kirby, M.M., Sherris, J.C. and
Turck, M. (1966). Antibiotic susceptibility
testing by a standard single disk method. American J. Clinical Pathology, 45:493-496 Leibovici, L., Paul, M., and Andreassen, S. (2010). Balancing the benefits and costs of
antibiotic drugs: the TREAT model. Clinical
Microbiology and Infection. 16: 1736–1739 Betoni, J.E., Mantovani, R.P., Barbosa, L.N., Di
Stasi, L.C.,and Fernandes, J.A. (2006). Synergism between plant extract and
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Presentation Australian Society for Micro-
biology Annual Scientific Meeting 2014 Patrick, Muller., Dagmar, G. Alber., Lynne,
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Cynthia,
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165–173 Taormina, P.J., Niemira, B.A., andBeuchat, L.R. (2001). Inhibitory activity of honey against
foodborne pathogens as influenced by the
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27, 238–254 Wilkinson, J.M., and Cavanagh, H. M. (2005). Antibacterial activity of 13 honeys against
Escherichia
coli
and
Pseudomonas
aeruginosa. J Med Food, 8:100 -103 ARABIC SUMMARY التأثيرات التآزرية للعسل والمضا
دات الحيوية المستخدمة عادة على
البكتريا موجبة الجرام ھدفت
ھذه
الدراسة
إلى
تقييم
ا
لتأثير
المثبط
لكل من ع
سل السدر وعسل السمور منفردين وكذلك تقييم التأثيرات
التآزرية لكل منھما مع 5 مضادات حيوية على ا
لبكتريا موجبة الجرام
. وتعد ھذه
ھي المرة األولى التي يتم فيھا تقييم
التأثير
المثبط والتأثير التآزري
لعسل السمور
على البكتريا
. أظھرت النتائج أن كال من عسل السدر وعسل
السمور منفردين لھما
تأثير مثبط على المعزوالت اإلكلينيكية والمعزوالت المرجعية لميكروبي االستافيلوكوكس
أوريوس
واالستربتوكوكس
بيوجين
، وكانت المعزوالت المرجعية أكثر حساسية لعسل
السدر بينما
كانت ال
معزوالت اإلكلينيكية أكثر حساسية لعسل
السمور
. بينت الدراسة أن الزيادة في تركيز العسل منفردا أو مع المضادات الحيوية أدى إلى زيادة التأثير المثبط ع
لى
البكتريا
. وقد شوھد أعلى تأثي
ر تآزري للعسل مع المضادات الحيوية
على ميكروب االستربتوكوكس بيوجين
حينما تم
استخدام
عينتي العسل مع األوفلوكساسين،
الببراسيللين، األموكسيسيللين
+
الكالفيوالنيك أسيد
والسلفامثوكسازول
+
التراميثوبريم
. أثبتت الدراسة أن استخدام عسل السدر وعسل السمور يحسنان نشاط المضادات الحيوية ضد البكتريا موجبة
الجرام
.
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Adenosarcoma pT3a TNM Finding v7
|
Definitions
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cc-by
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Adenosarcoma pT3a TNM Finding v7 National Cancer Institute Qeios ID: EOVFOH · https://doi.org/10.32388/EOVFOH Qeios · Definition, February 8, 2020 Open Peer Review on Qeios Source National Cancer Institute. Adenosarcoma pT3a TNM Finding v7. NCI Thesaurus. Code
C89619. Tumor involves abdominal tissues, one site. (from AJCC 7th Ed.) Qeios ID: EOVFOH · https://doi.org/10.32388/EOVFOH 1/1
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Editorial: Next generation γδ T cell-based tumor immunotherapy
|
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Andy Hee-Meng Tan 1*, Alice Man Sze Cheung 2,3*
and Dieter Kabelitz 4* 1Immune Cell Manufacturing, Bioprocessing Technology Institute Agency for Science, Technology
and Research (ASTAR), Singapore, Singapore, 2Department of Hematology, Singapore General
Hospital, Singapore, Singapore, 3SingHealth Duke-National University of Singapore (NUS) Medicine
Academic Clinical Programme, Singapore, Singapore, 4Institute of Immunology, University of Kiel,
Kiel, Germany gamma delta (gd) T cells, tumor immunotherapy, butyrophilins, cytokine
programming, gamma delta TCR repertoire, cost-effective manufacturing of gamma
delta t cell therapies, chimeric antigen receptor-engineered gamma delta T
cells, phosphoantigens Editorial on the Research Topic
Next generation gd T cell-based tumor immunotherapy Editorial on the Research Topic
Next generation gd T cell-based tumor immunotherapy COPYRIGHT
© 2022 Tan, Cheung and Kabelitz. This
is an open-access article distributed
under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction
in other forums is permitted, provided
the original author(s) and the
copyright owner(s) are credited and
that the original publication in this
journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is
permitted which does not comply with
these terms. The discovery of the gd T-cell receptor (TCR) and its ability to confer potent
cytotoxic activity in CD3+ cells some 35 years ago sparked the initial proliferation in
research that garnered widespread interest in the biology and function of gd T cells. The
identification of major human gd TCR clonotypes, their tissue distributions as well as
dynamic changes throughout ontogeny and in disease states have contributed to the
appreciation of human gd T cell diversity. Despite this, incomplete understanding of
mechanisms underlying the complexity of various gd T cell subsets in homeostasis,
inflammation and malignancy restricted the focus of most early studies to blood
circulating Vg9Vd2 T cells which could be robustly activated and expanded ex vivo
using aminobisphosphonates such as zoledronate compared with other gd T cell subsets
for which activating ligands were then largely unknown. This galvanized attempts to
harness the tumoricidal potential of Vg9Vd2 T cells in clinical trials. Although these cells
exhibited highly promising safety profiles in patients, early trial data revealed suboptimal
anti-tumor efficacy. Despite these setbacks, recent breakthroughs in deciphering the
unique antigen (Ag) binding modes of gd TCR coupled with high dimensional analyses of
tissue- and disease-specific gd T cell subsets at single cell resolution led to renewed
excitement in the development of gd T cell-based therapeutics. TYPE Editorial
PUBLISHED 30 September 2022
DOI 10.3389/fimmu.2022.1041362 TYPE Editorial
PUBLISHED 30 September 2022
DOI 10.3389/fimmu.2022.1041362 Editorial: Next generation
gd T cell-based tumor
immunotherapy OPEN ACCESS
EDITED AND REVIEWED BY
Katy Rezvani,
University of Texas MD Anderson
Cancer Center, United States
*CORRESPONDENCE
Andy Hee-Meng Tan
andy_tan@bti.a-star.edu.sg
Alice Man Sze Cheung
alice.cheung@duke-nus.edu.sg
Dieter Kabelitz
Dietrich.Kabelitz@uksh.de
SPECIALTY SECTION
This article was submitted to
Cancer Immunity
and Immunotherapy,
a section of the journal
Frontiers in Immunology
RECEIVED 10 September 2022
ACCEPTED 20 September 2022
PUBLISHED 30 September 2022
CITATION
Tan AH-M, Cheung AMS and
Kabelitz D (2022) Editorial: Next
generation gd T cell-based
tumor immunotherapy. Front. Immunol. 13:1041362. doi: 10.3389/fimmu.2022.1041362
COPYRIGHT
© 2022 Tan, Cheung and Kabelitz. This
is an open-access article distributed
under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction
in other forums is permitted, provided
the original author(s) and the
copyright owner(s) are credited and
that the original publication in this
journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is
permitted which does not comply wit
these terms. OPEN ACCESS EDITED AND REVIEWED BY
Katy Rezvani,
University of Texas MD Anderson
Cancer Center, United States Andy Hee-Meng Tan 1*, Alice Man Sze Cheung 2,3*
and Dieter Kabelitz 4* Andy Hee-Meng Tan 1*, Alice Man Sze Cheung 2,3*
and Dieter Kabelitz 4* This Research Topic has
compiled a series of nine articles of which five review our hitherto understanding of the
multifaceted nature of gd T cells and four report original research providing new insights
into the molecular and cellular regulation of a diverse repertoire of gd T cells, paving the
way for next generation gd T cell-based tumor immunotherapies. Frontiers in Immunology frontiersin.org 01 Tan et al. 10.3389/fimmu.2022.1041362 Despite extensive use of phosphoantigens (pAgs) to activate
and expand Vg9+Vd2+ T cells, the role of butyrophilins (BTNs)
in mediating Vg9Vd2 TCR-dependent activation is only gaining
recent appreciation. Expression of BTN3A1 and BTN2A1
heterodimers, in complex with pAgs, on the surface of
accessory innate immune, infected or tumor cells is an
absolute requirement for interaction with the Vg9Vd2 TCR. Other BTN and BTN-like (BTNL) molecules are involved in the
ontogeny and homeostasis of non-Vg9+Vd2+ T cell subtypes as
noted by Herrmann and Karunakaran. Their review also
emphasized dissection of BTN-associated mechanisms that
enhance the effector function to broaden the therapeutic
applications of gd T cells in disease settings. Extending the role
of TCRd complementarity-determining region 3 (CDR3d) in
Vg9Vd2 TCR-mediated responses, Vyborova et al. adduced
evidence for CDR3d determinants in pAg sensing that are
significantly correlated with Vg9Vd2 TCR affinity and signal
strength, contributing to Vg9Vd2 T cell repertoire focusing. Furthermore, surface expression of the inhibitory natural killer
receptor (NKR) CD94/NKG2A is biased toward while that of
activating NKR NKG2D is independent of these CDR3d traits,
findings which may impact design of high-affinity Vg9Vd2 TCR-
based therapies. model. Hu et al. examined the immunosuppressive TME of
hepatocellular carcinoma (HCC) which the authors showed close
correlation with high expression of inhibitory checkpoint
molecules, presence of tumor-promoting immune cells and
various types of programmed cell death. This is associated with
gd T cell subtype imbalance characterized by selective depletion of
cytotoxic Vd2+ T cells and enrichment of Treg-like Vd1+ T cells as
well as poorer patient prognosis. These studies underscore the
importance of performing greater in-depth analyses of the cellular
networks and interplay of cytokines in the TME to accelerate the
clinical translation of gd T cell therapies. Over the years, translating gd T cell therapies from bench to
bedside has encountered tremendous challenges. Saura-Esteller
et al. expertly summarized the evolution of gd T cell therapeutic
strategies assessed in clinical trials that signifies past progress in
gd T cell research. Andy Hee-Meng Tan 1*, Alice Man Sze Cheung 2,3*
and Dieter Kabelitz 4* With a growing list of companies developing
gd T cell-based or engaging therapies, the authors noted the high
cost of manufacturing gd T cell-based products due in part to
requirement for multiple cytokines and prolonged ex vivo
expansion process. Exemplifying continual efforts to reduce gd
T cell production cost, Ferry et al. described a one-step protocol
utilizing a single cytokine to expand Vd1+ T cells. In addition,
the authors demonstrated that these cells were amenable to high
efficiency transduction of chimeric antigen receptors (CARs). Going forward, development of other ways that harness the anti-
tumor potency of gd T cells, namely bispecific gd T cell engagers
and advanced gd T cell genome-editing, is expected to widen the
immuno-oncological applications of these cells. p
gd T cells manifest substantial plasticity and can be polarised to
different cell states in response to environmental stimuli. It is thus
not uncommon to identify gd T cells with opposing functional
properties in different biological contexts. The mini review by Bhat
et al. provides a comprehensive summary of studies highlighting
the dichotomous nature of gd T cells influenced by diverse
pathological milieux, leading to beneficial or detrimental
outcomes in the host. For example, IL-17 production by gd T
cells has been shown to aggravate autoimmune conditions (1, 2)
and also be associated with immunosuppression that exacerbate
tumour growth (3, 4). Such phenomena attributable to the varied
cellular interactions of gd T cells can be addressed by appropriate
considerations of systems immunology and personalized
approaches. In this regard, Chan et al. provided an updated
account of crosstalk between gd T cells and a variety of immune
cells which collectively coordinates anti-tumor responses. Additionally, the cytokine milieu plays a major role in shaping
the fate commitment of gd T cells. Consistent with this, Song et al. discussed how cytokines and their combinations differentially direct
the polarization of tissue-resident and tumour-infiltrating gd T cells. Such experimental insights will inform strategies to manipulate the
cytokine dependencies of gd T cells to improve the cytotoxic
function and in vivo persistence of administered T cells against
tumors. Pei et al. elegantly showed that CD137 costimulation of Vg9
+Vd2+ T cells using CD137L agonist diminished IL-10 receptor
expression and alleviated exhaustion in these cells by mitigating the
immunosuppressive tumor microenvironment (TME) mediated by
IL-10. This increased Vg9+Vd2+ T cell efficacy against Epstein Barr
virus (EBV)-transformed B lymphoma in a humanized mouse Funding This work is supported by the core funds of BTI, A*STAR
and Industry Alignment Fund-Industry Collaboration Projects
(IAF-ICP) grant ID I1801E0037 awarded to AH-MT. AMSC is
supported by National Medical Research Council (NMRC) of
Singapore-Centre Grant NMRC/CG21APR2002. Author contributions AT and AC discussed findings of various articles of the
Research Topic in broader context of tumor immunotherapy. AT collated contributions and prepared final version of editorial
by taking into account suggestions from AC and DK. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Frontiers in Immunology frontiersin.org 02 10.3389/fimmu.2022.1041362 10.3389/fimmu.2022.1041362 Tan et al. Publisher’s note organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher. All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated 3. Wu P, Wu D, Ni C, Ye J, Chen W, Hu G, et al. gdT17 cells promote the
accumulation and expansion of myeloid-derived suppressor cells in human
colorectal cancer. Immunity (2014) 40(5):785–800. doi: 10.1016/
j.immuni.2014.03.013 4. Peng G, Wang HY, Peng W, Kiniwa Y, Seo KH, Wang R-F. Tumor-
infiltrating gd T cells suppress T and dendritic cell function via mechanisms
controlled by a unique toll-like receptor signaling pathway. Immunity (2007) 27
(2):334–48. doi: 10.1016/j.immuni.2007.05.020 2. Petermann F, Rothhammer V, Claussen MC, Haas JD, Blanco LR, Heink S,
et al. gd T cells enhance autoimmunity by restraining regulatory T cell responses via
an interleukin-23-Dependent mechanism. Immunity (2010) 33(3):351–63.
doi: 10.1016/j.immuni.2010.08.013 References 1. Sutton CE, Lalor SJ, Sweeney CM, Brereton CF, Lavelle EC, Mills KHG. Interleukin-1 and IL-23 induce innate IL-17 production from gd T cells, amplifying
Th17 responses and autoimmunity. Immunity (2009) 31(2):331–41. doi: 10.1016/
j.immuni.2009.08.001 2. Petermann F, Rothhammer V, Claussen MC, Haas JD, Blanco LR, Heink S,
et al. gd T cells enhance autoimmunity by restraining regulatory T cell responses via
an interleukin-23-Dependent mechanism. Immunity (2010) 33(3):351–63. doi: 10.1016/j.immuni.2010.08.013 03 Frontiers in Immunology frontiersin.org 03
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German
| null |
Ueber die Hungerempfindung<sup>1</sup>)
|
Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift
| 1,915
|
public-domain
| 6,270
|
Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Aus der Medizinischen Poliklinik der Universitht in Würzburg. Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. g
p
g
g
Die geschilderten Hungerempfindungen treten aber auch
entschieden zurück, wenn die Zeit der gewohnten Nahrungs-
aufnahme überschritten ist, sie stellen sich erst gegen Abend,
zur Stunde des Abendbrotes, erneut und nun um so heftiger
wieder ein. Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. LJeber die Entstehung des Hungergefüliles hat man sich
iiocli nicht viel Gedanken gemacht. Die Sache liegt scheinbar
gamjz einfach: die ,,Lcere des Magens" ist es, welche zur Aus-
lösung des Hungers führt. Jm nun die Frage zu entscheiden, ob die Füllung
des Nagens als solche den Hunger zu stillen vermag,
nahm Herr Dr. Timorna, naandem er von 7 Uhr morgens
gehungert hatte, um 2 TJhr nachmittags einen halben Liter
dicken Baryumbreies. 1)araufhin schwand sofort das
Gefühl der Spannung und des Nagens in der Magen-
grube um nach drei Stunden freilich unvermittelt und um
so heftiger wieder aufzutreten. Das Gefühl der auge-
meinen Schwäche und der Hinfälligkeit verschwand
aber nicht. Es blieb vielmehr unbeeinflußt bis zur Auf-
nahme einer reichlichen Abendmahlzeit bestehen. Ausrh ein
junger Kollege, Herr Cand. med. Dengg, der sich demselben
Versuch unterwarf, gab an, daß nach der Aufnahme des Baryum-
breies zwar die örtlichen Empfindungen in der Magengegend
schwanden. daß er sich aber nicht gesättigt fühlte. Schon
zwei Stunden danach, zu einer Zeit also, in der bei der Röntgen-
untersuchung noch Baryum im Magen nachweisbar ist, trat
wieder heftiger Hunger mit dem Geflihl der Spannung in der
Magengrube auf. Manche Forscher, nie z. IB. H a 1er, nahmen ai'. daß die Wan-
(lungen des leeren Magens sieh heriihren und daß durch dic Reibung
und damit durch
die ,Spannung und Zerrung der Nagennerven" die
Hungerenipfindung entstiiiide. Andere Autoren. wie z. B. I)arwi n,
glaubten, daß der Ieeae Nagen erschlafte irnd daß so der Hunger zu-
ste fldekoni inc. Wieder andere vermn uten, da B die
erniehrte Turgeszenz
der Magenwiinile oder daß die Absonderung der Salzshure und eine bi-
innendc Selbstverdauung der Magenschleimhaut der H ungerein pfind nog
zugrundeliegen
Alle diese Autoren sprecher< den Magen als den Ent-
stehungsort (les Hungers ei<. Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Auch Sternberg, dein w ir eine Reihe
von literarischen Studien über das Problem <les Appetits verdanken
<«<cl der alle diese Arbeiter< j iingst in einem Buche
.T)as Nabrungs-
bedürfnis, der Ap1stit und <1er [lunger''2) zusammenfailte. erlegt die
Entstehung der Hungen-nipfinilung in den Magen-.,. das Ilungeigefiihi
tritt nur dann auf. u cnn der Magen leer iSt." SchlieLllich vermutet auch
Krehl in seiner ,, Pathologiscliemi Physiologie des Menschen'', daß der
hingen der Ort se], an a eichen< das Hiiogergefùhl eiitstehe, und stützt
seine Meinung <nit tier Becbaehtung, da LI sieh emi<e Steigerung <1<-s H iii<g('r-
gelühls in manchen Frihleii findet, in welchen sich der Mager< rasch< cnt -
leert. Neisser und Bräuning ') glauben durch Versuche fest.'
gestellt zu haben, daß bei einem konstanten intragatralen
Druck vomi 16-18 cm H2O normales Sättigungsgefühl auftz'ete
. Dieser Druck wird erzeugt durch die ,,peristolische" Kón-
traktion des Magens einerseits und die Masse der Nahrung
anderseits". Durch Schnüren kann man nach Weisser und
Bräuning den Druck im Nagen erhöhen und dadurch ein
vorzeitiges Gefühl der Sättigung hervorrufen. Da umicli nun all
diese Erklärungen und Vemnimitungemi
nicht befriedigten, veranlaßte ich einige jüngere Kollegen, (lie
frillier am Städtischen Krankenhause in Augsburg und nun
in Wiirzhurg mit mir zusammenarbeiteten, diese Fragen ex-
perimentell in Angriff zu nehmen. Herr Dr. Timoma, welcher
besonders eifrig und besonders aufopferum<gsvoll Selbstversuche
vornahm, verarbeitete dann das gesammelte Material zu einer
Thssertation: Eine Studie über die Hungerenipfindung,"
B
i h
d
E
b i
ll Auf Grund unserer Versuche sind wir zu der Uebes'zeugung
gekommen, daß die Füllung des vorher leeren Magens mit unre-
sorbierbarer Kost nur den Teil der Hungerempfindtmngen, wel-
ehen wir in die Magengegend verlegen, stillt. Und auch diesen
nur vorübergehend. Die Füllung des Magens mit Wasser oder
mit leerer Suppe nimmt uns wohl den örtlichen Druck in der
Magengrube, nicht aber den Hunger. Davon, daß eine Füllung
des Magens als solche die Sättigung" bedingt, kann wohl keine
Rede sein . Sind wir doch nach längerem Fasten oder nach an-
strerigender körperlicher Leistung trotz Füllung de
Magens
noch nicht satt. b i h
d
i
U
h
d Bevor ich nun das Ergebnis all dieser Versuche kurz
zusammenfasse, seien clic Empfindungen besprochen, die nach
längerer Nahrungsenth altung auftreten Wird «ach einem Frühstück in den Morgenstunden (um 7
TJhir'
a. Begründet von Dr. Paul Börner VERLAG:
GEORG THIEME
LEIPzIG
Antonstraule IS HERAUSGEBER
Geh. San.- Rat Prof. Dr. Schwalbe
Berlin-Charlottenburg, Schlüterstr. 53 41. JFIHRQIiNG 1) Ein kurzem Bericht über diese schon vor längerer Zeit vor-
genonunenen Untersuchungen, wurde auf dciii südwestdeutsehen Neuro-
logentag in Baden-Baden erstattet.
2) Leipzig 1913. 2) Deber normale und über vorzeitige Sättigung.
M. mn. W. 1911
Nr. 37.
2) W. ki. W. 1893 Nr. 31. Nr. 44 Dieses Dokument wurde zum persönlichen Gebrauch heruntergeladen. Vervielfältigung nur mit Zustimmung des Verlages. oder körperliche Tätigkeit in Anspruch genommen, so kommen
diese Hungerempfindungen viel weniger zum Bewußtsein. i
hild
fi d
b
h oder körperliche Tätigkeit in Anspruch genommen, so kommen
diese Hungerempfindungen viel weniger zum Bewußtsein. 41. JFIHRQIiNG Nr. 44 Ddllisdhe Nedizillisthe Wochefischrift Begründet von Dr. Paul Börner Begründet von Dr. Paul Börner Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. So können Kranke, bei denen die Entleerungsfähigkeit
des Magens durch eine Pförtuerverengerung beeinträchtigt ist,
trotz gefüllten Magens lebhaften Hunger empfinden. d
i
b di
di
d
d
h Anderseits bedingt die Leere des Magens durchaus
nicht immer Hunger. ist doch erwiesenermaßen 2-3 Stunden
nach der Aufnahme des Frühstücks der Magen wieder leer,
die Hungerempfindung stellt sieh aber erst 4-5 Stunden nach
dem Frühstück wieder ein. Auch morgens beim Aufstehen
ist der Magen des Gesunden sicher leer, trotzdem besteht
kein Hunger, zum Nahrungsbedürfnis kommt es erst etwa
eine halbe bis eine Stunde danach. Der Magen kann also nicht die Stelle sein, von
der das Bedürfijis nach Nahrungsaufnahme aus-
gelöst wird. Das mag auch daraus geschlossen werden, daß
Leute, denen der Magen wegen ausgebreiteter Geschwulst-
bildung zum größten Teile oder fast ganz herausgenommen
wurde, bei Nahrungsenthaltung gerade so ITunger empfinden
wie vordem in gesunden Tagen. Ja, nach Perth es 1) soll gerade
nach Querresektion des Magens das Nahrungsbedürfnis be-
sonders lebhaft sein können. fibs würde aber nicht der Fall
sein, wenn der Magen wirklich der F4ltstehungsort des Hungers
ware. Wenn nun der Magemi als Entstehungsort des Hunger-
gefishis nicht in Betracht kommt und wenn audi die Vermutung,
daß der hunger ein Ahlgemeingefühl ist, das ini ganzen Körper
entsteht, nicht begründet werden kann, auf .welchem Weg
kommt es uns dann zum Bewußtsein, daß der Körper der
Nahrungszufuhr bedarf? g
Einen Hinweis für die Erklärung der Entstehung des
Hungers liefert uns vielleicht die Physiologie der Luft-
11 ungere mpfindung. Als Beweis für die Behauptung, daß der Magen der Ort
sei, von dem der Hunger ausgelöst wird, führen nun manche
Autoren die Tatsache an, daß das Hungergefühl durch die
Einnahme von Kokain per os gestilit werden könne. J)er Lufthunger wird erwiesenermaßen nicht dort aus-
gelöst, wo er empfunden wird. Nicht die mangelnde Ventilation
der Lungen liegt der in den Brustkorb lokalisierten Atemnot
und der Beengung dort zugrunde; die erhöhte Venosität des
Blutes ist es vielmehr, weiche das im oberen Kopfmarke be-
findliche Atemzentrum reizt und von dort aus die stürmischen
Atem bewegungen verursacht. So behauptet A. Valenti 2), durch Kokainisicrung des Pharynx
oder der Magensehiehuhaut oder des Vagus (! ) den Hunden jedo FreB-
lust genommen zu haben. Auch H. Schlesinger glaubt ,,durch An-
ästhesierung auch nur eines Teiles der Magenschleimhaut für die Dauer
der örtlichen Einwirkung des Mittels das Hungergefühl wesentlich
herabgesetzt zu haben". Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Da man nun einwerfen kann, dal3 im letzteren Versuch
die Fffllung des Darmes das Hungergefühl stille, so veran-
laBte ich Herrn Dr. Thoma, den Magendarmkanal als Re-
sorptionsstelle ganz
auszuschalten und
sich parenteral,
durch subkutaiie Injektion, Nahrung zuzuführen. j
g
Als sich urn die Mittagsstunde lebhafter Hunger einstellte und
al
es dann au lautem Kollern und Gurrcii in der Magengegend ge-
kommen war, ließ sich Dr. Tho ma einen Liter einer 6 % igen Trauben-
zuckerlösung subkutan infundieren. Die Hungerempfindung, d. h. so-
wohl der lokale Druck in der Magengegend als auch die Ailgenicin-
erscheinungen, wie die korperliche Hinfälligkeit und die Müdigkeit
ließen daraufhin
ntschieden nach. g
Die anfänglich erwühnte Theorie, es sei die ausgeschiedene
S a I z s ä u r e , welche die Humigerempfindung in der Magen-
gegend auslöse, konnten wir dadurch leicht widerlegen, daß
wir in den Vormittagsstunden zu einer Zeit, in welcher das
Frühstück den Magen schon verlassen hatte, in der aber noch
kein Hunger bestand, Salzsdure einnahmen. Es gelang uns
aber dadurch nicht , Hungerempfindung auszulösen. Dieses Dokument wurde zum persönlichen Gebrauch heruntergeladen. Vervielfältigung nur mit Zustimmung des Verlages. Freilich stellte sich in all diesen Versuchen bald wieder
Hunger ein, denn die Nalirungsmengen, welche durch ein
Klisma oder auf parenteralem Wege zugeführt werden können,
sind für einen gesunden, körperlich und geistig arbeitenden
Menschen immer ungenügend. Gegen die allgemein gültige Annahme, daß die Leere
des Magens es sei, welche der Hungerempfindung zugrunde-
liege, lassen sich nun audi eine Reihe von klinischen Beob-
achtungen ins Feld führen. g
p
g
Ueber die schwierig zu beantwortende Frage, wie uns
denn das Bedürfnis nach Nahrungsaufnahme zum Bewußtsein
kommt, helfen sich manche Autoren dadurch hinweg, daß
sie den Hunger als ein Ahlgemeingefühl, das im ganzen
Körper entsteht, ansprechen. Zweifellos leiden nun unter
dem Nahrungsmangel sämtliche Zellen sämtlicher Organe und
sämtlicher Gewebsarten. Zum Bewußtsein kann uns aber
diese Unterernkärung der Gewebe doch nur dann kommen,
wenn nervöse,
zentripetal leitende Verbindungen zwischen
diesen Zellen und unserem Geh irne bestehen und wenn auf diesem
Wege spezifische sensible Erregungen von den an Nahrungsnot
leidenden Zellen nach dem nervösen Zentralorgane gelangen. Nun ist der Nachweis von solchen Bahnen nicht erbracht und
auch wohl nicht zu erbringen. Und so wird audi mit dem
Schlagwort : der Hunger ist ein Allgemeingefiihl" das Ver-
ständnis für (lie Entstehung der Hungerempfindung nicht ge-
fördert. Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. rn), das aus einer Tasse Milch und einem Brutcheri besteht, keine
weitere Nahrung mehr aufgenommen, so stellt sich meist uni clic Mittags-
stunde, also etwa uni
12 Vhr, unter kollernden und gurrenden Ge-
rätischen im Epigastriuni ein leichtes D rue kgefühl iii der Magen-
gegend ein. Dieses wird vielfach als ein Gefühl der Leere'', der ,,Oede"
«der des Nagens" bezeichnet. . In gleichem Zeit kommt es aueh zu
verinehrter Speicbelahsonilerung,
zum Schluckreiz und zum
Schluekbcdürfnis und manchmal auch zum Gähnen. Verabreicht man anderseits unter Umgehung des
Magens eine reichlichere Menge rasch resorbierbarer Nähr-
substanz, so schwinden sowohl das örtliche Gefühl in der
Magengegend als auch die allgemeine körperliche und geistige
Hinfälligkeit! Die Versuche wurden von Herrn Dr. Thom a
iii der Weise angestellt, daß er sich um die Mittagszeit, ala
er lebhaften Hunger empfand, ein Nährklisma mit Trauben-
zucker und Erepton (abgebautes Eiweiß) verabreichte. Iinterläßt
mitan trotz dieser Mahnungen es nun, Nahrung auf-
zunehmen, so gesellt sieh
bald ein Gefuhl der körperlichen Schwäche
(,,Magenschwäche") nnd der geistigen Ermüdung dazu. Es besteht
ein ausgesprochenes Vnlustgefühl. Schließlich kann es bei längerer
Nalirungsenthaltung zum
Schwindel, zu Flimmern vor cIen Augell,
zum Ohrensausen und endlich zur wirklichen Erschiipfung kommen. Das örtliche Gefühl der Leere in der Magengegend ließ
freilich nicht völlig nach, volil aber verschwand die EmpfIndung
der körperlichen und geistigen Hinfälligkeit. Aehnlmohe Unter. suchungen nahm if. Schlesinger 2) schon im Jahre 1893 All diese - angefiihrten Empfindungen sind in der Ruhe
und je mehr niali Gelegenheit hat, sie 'zu beachten, stärker. ist man dagegen durch irgendwelche anstrengende geistige 173 DEUTSCHJE MZDIZINJSCHE WOCRENSCBmJFT. 1298 Nr. 44 vor. Auth seine Versuche ergaben, daß eine Stillung des
}iungergefühles durch eine nicht vom Magen aus erfolgende
Nahrungszufuhr möglich ist". des Kokairis auf das Zentralnervensystem es sein, welcher die
durch den Mangel an Nahrungsaufnahme entstehenden un-
angenehmen Empfindungen zurückdrängt. Das Bestehen von
sensiblen Endorganen in der Magenschleimhaut, auf
welche das Kokain anästhesierend wirken könnte, ist bisher
weder auf anatomischem, noch auf physiologisehem oder kil-
nischem Wege festgestellt worden. Im Gegenteil, es ist ganz
unwahrscheinlich, daß der Magen s e h lei m h a ut irgendeine Art
von Sensibilität zukommt
Schließlich gibt es auch andere
Stoffe, welche den Hunger vertreiben können, wie das Nikotin
odér wie starker Tee
Aber auch hier kommt nicht Betäubung
sensibler Nerven in der Magenschleìmhaut als vielmehr Beein-
flussung des Gehirns in Betracht. î) D. Zsehr, f. Chir. 12ü. - 2) Bolleltino itelle Soejeta Medico-
Chirurgica di Pavia 1909.
3) Arch, f. Verüainmgskr. 21. Fehr. 19l. Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Und wir gehen wohl nicht irre, wenn
wir vermuten, es möchte audi die SteHe, von der die Nahrungs-
aufnahme reguliert wird, im Zwischenhirn und nahe den Wan-
dungen des dritten Ventrikels gelegen sein. Auch der von R o th manu ) seines Großhirns beraubte Hund
konnte saufen
Allmhhlich kehrte auch die Fähigkeit, feste Nahrung
aus dem Napfe zu nehmen, wieder. Auch ein Fisch oder ein Wurm hat Hunger. Sie fressen
so
lange,
bis das Nahrungsbedürfnis gedeckt
ist. Auch
bei diesen Tieren müssen gewisse Zeligruppen des Zentral-
nervensystems durch den Nahrungsmangel gereizt werden, die
dann die Sucht nach Nahrungsaufnahme und die Freßreflexe
auslösen. Eine solche Vermutung wurde schon im Jahre 1914 von deni
Würzburger Psychiater R ei cha rd t ausgesprochen. R ei chard t weist
darauf hin, daß im Zwischenhirn ein ,,hypothetisches vegetatives Zentrum
f ür das Körpergewicht und den Stogwechsel" zu suchen ist und dali
man , das Hunger- und Durstgefühl zum Teil mit einer Erkrankung
dieses Zentrums in Verbindung bringen darf"1). Unter Reicliardts
Leitung st von Dreseher eine 1)issertation ,,Ueber die Störung des
Hunger- und Durstgefühles bei Hirnkrankheiten"') erschienen. Dieseher
stellt aus der Literatur zahlreiche Beobachtungen zusammen, in denen
es im Anschluß an Erkrankungen der Hypophyse zu Störungen des
l)urst- und des Hungergefühles gekommen war. Besonders interessant
sind die Fälle von Akromegahie, die mit Polyphagie mid Polydipsie
cinhergingen. Mit Recht weist Dreseher darauf hin, daß die Frage
der hypophysären Fettsucht und der hypophysären Bulimie noch nicht
einwandfrei im positiven, d. h. innersekretorischem, Sinne zu beant-
worten ist, denn es läßt sieh nicht mit Sicherheit ausschließen, ,,ob
nicht doch Teile der Infundibulargegend oder andere Teile des Zwischen-
liirnes selbst erkrankt waren". Die Stellen de
Zentralnervensystems, von welchen alle
diese Jebenswichtigen vegetativen Funktionen reguliert werden,
sind bei uns und bei allen Wirbeltieren an dem gesehütztesten
Orte des ganzen Körpers gelegen. lije Großhirnhemisphären, das Kleinhirn und gar das
Rückenmark sind viel mehr Schädigungen ausgesetzt als die
mittleren Partien der Basis des Gehirns. Kommt ès aber ein-
mal, wie dies bei Operationen an der Hypophyse der Pall
sein kann, zu einer Verletzung des Infundibulums oder der
Wandungen des dritten Ventrikels, so stellt sich meist rasch
der Tod ein. Freilich sind wir nicht in der Lage, genauer die Stelle
bestimmen zu können, von welcher aus die Regulierung der
Aufnahme der Flüssigkeit und der Aufnahme der festeren
Nahrungsstoffe erfolgt. Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. ll
il
i
i d ß
h
i
b Dieses Dokument wurde zum persönlichen Gebrauch heruntergeladen. Vervielfältigung nur mit Zustimmung des Verlages. g
Kollege Faust teilt mir mit, daß nach seinen Beob-
achtuiigen Tiere, bei denen der Würmestich einwand-
frei gelungen ist, auch regelmäßig jede Freßlust
verl ieren. g
Kollege Faust teilt mir mit, daß nach seinen Beob-
achtuiigen Tiere, bei denen der Würmestich einwand-
frei gelungen ist, auch regelmäßig jede Freßlust
verl ieren. Daß dies nicht der Fall sein kann, das geht auch aus den Beob-
achtungen von Goltz ') hervor, die dieser Physiologe an Hunden, denen
er das Großhirn herausgenommen hatte, machte. Ein solcher Hund
ging dann, wenn er längere Zeit nicht gefüttert worden war, unruhig
un Käfig umher und ließ die Zunge aus dem Maule hängen. Zu Leek-
bewegungen gesellten sich Kaubewegungen. Hielt man dem Hunde
eine Schüssel Milch vors Maul, so begann er sofort zu saulen. Nach
der Nahrungsaufnahme wurde er wieder ruhig und legte sieh zum Schlafe. Daß dies nicht der Fall sein kann, das geht auch aus den Beob-
achtungen von Goltz ') hervor, die dieser Physiologe an Hunden, denen
er das Großhirn herausgenommen hatte, machte. Ein solcher Hund
ging dann, wenn er längere Zeit nicht gefüttert worden war, unruhig
un Käfig umher und ließ die Zunge aus dem Maule hängen. Zu Leek-
bewegungen gesellten sich Kaubewegungen. Hielt man dem Hunde
eine Schüssel Milch vors Maul, so begann er sofort zu saulen. Nach
der Nahrungsaufnahme wurde er wieder ruhig und legte sieh zum Schlafe. Auch der von R o th manu ) seines Großhirns beraubte Hund
konnte saufen
Allmhhlich kehrte auch die Fähigkeit, feste Nahrung
aus dem Napfe zu nehmen, wieder. Ed. Aronsohn, dein wir ja die ersten Arbeiten über das
wärmeregulierende Zentrum im Zwischcnhirn verdanken, stellte
fest, daß ein Bluterguß in den dritten Ventrikel bei den Ver-
suchstieren jedesmal zum Nachlassen der FreBlust geführt habe. S hli ßli h d
h di
ll
li h
h
d B
b Ed. Aronsohn, dein wir ja die ersten Arbeiten über das
wärmeregulierende Zentrum im Zwischcnhirn verdanken, stellte
fest, daß ein Bluterguß in den dritten Ventrikel bei den Ver-
suchstieren jedesmal zum Nachlassen der FreBlust geführt habe. Schließlich deutet auch die alltäglich zu machende Beob-
achtung, daß mit dem Fieber eine Beeinträchtigung der Hunger-
empfindung, des Appetits, einhergeht, auf eine nahe Beziehung
de
Regulationszentrums für die Körperwärme und für die
Nahrungsaufnahme. 3) zitiert nach A. Sohühler; Die Erkrankungen der Zirbeldiüse.
Handbuch der Neurologie, von Lewandowsky 4. 5) Arbeiten aus der Psychiatrischen Klinik in Würzburg. Jena
1914 8. S. 690.
2) Würzburg 1913
3) i i
h A S hühl
Di E k
k
d
Zi b ldiü Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Alle diese Vermutungen wären nun wohl zu
begründen, wenn dem Magen wirklich, wie das B. Stiller noch in einer
vor kurzem erschienenen Arbeit) annimiit, eine ,,spezifische Sensibi-
lität" zukänse, wenn der Hunger wirklich ,,in den Magennervcn" ent-
stehen würde. Für eine solche Annahme fehlen uns aber, wie oben dar-
gelegt, die Beweise. So lange dem Kind im Mutterleib durch die Nabelschnur
sauerstoffreiches und nähirstoffhaitiges Blut zugeführt wird,
kommt es trotz luftleerer Lungen und trotz leeren Magens
weder zur Atemnot noch zum Hunger
ist diese Zufuhr aher
unterbunden, so stellt sich nach Aufzehrung des Sauerstoffes
Atemnot und nach Verbrauch der vom mütterlichen Or-
ganismus noch mitgebrachten Nährstoffe Hunger ein. Dieser
geist mit sichtlichen TJnlustgefiihlen einher, die sich durch
klägliches Schreien und durch Saugbewegungen kundgeben
und die nur durch Nahrungsaufnahme gestilit werden. Durch Versuche an sich selbst haben nun Dr Tho ma, Dr. Markus
und Cand. med. Dengg nachgewiesen, deli (lurch Kokain nicht so sehr
die örtlichen Empfindungen in der Magengegend als vielniehr das all-
gemeine Gefühl der Müdigkeit und der Abspannung behoben werden. Das Gefühl der Schwäche und Erschöpfung maclit einen) solchen der
Anregung und der Frische Platz. lind zwar einerlei, ob Cocablätter
gekaut werden, ob Kokain per os genoni tiesi worden war, oder 01)
Kokain subkutan verabreicht wurde. Wir vermuten nun, daß, ähnlich wie der Mangel des Blutes
an Sauerstoff an einer bestimmten Stelle des Zentralnerven-
systems
die unlustbetonte Empfindung der Atemnot und
die Atembewegungen auslöst,
so auch der Mange] des
Blutes an abhaufähigen Substanzen von einer um-
sehiriehenen Partie des Gehirns die den Nahrungs-
hunger anzeigenden Empfindungen verursacht. Auf Grund dieser Versuche dürfen wir also wohl annehmen. daß nicht die Anästhesierung der Magenschleimhaut die Ur-
sache für die liungeastillende Wirkung des Kokains ist. Wir
müssen vielmehr vermuten, es möchte der erregende Einfluß g
g
p
g
Bei vermehrter körperlicher Arbeit stellt sich nicht nur
stärkerer Lufthunger, der sich in vertiefter und rascher Atmung
äußert, ein, im Anschin 13 au sie tritt auch der Nahrungshunger
in erhöhtem (rade auf. Der Magen kann nun nicht leerer DEUTSCHE MEDIZINISCHE WOOHENSOffitIFT. 1299 '28. Oktober 1915 '28. Oktober 1915 kontraktionen und dieMagendrüsen zur Tätigkeit angeregt. Unweit davon sind die Nuclei sahivatorii, die Ganglienzellün-
gruppen lokalisiert, von welchen die Speicheldrüsen innerviert
werden. In nächster Nähe sind auch die Ganglienzehlengruppen
gelagert, welche den Schluckreflexen vorstehen! als leer werden, und so deutet der Umstand, daLi nach körper
Ijeher Arbeit der Hunger zuiii;nint, darauf hin,
daI. ) Pflug. Arch. 51.
2) Verhandlungen der Gesellschaft deutscher Nervenärzte, Wien 1909. Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. es der
Mangel an Nahrungsstoffen ini Blute ist, der die Hunger-
empfindung auslöst. als leer werden, und so deutet der Umstand, daLi nach körper
Ijeher Arbeit der Hunger zuiii;nint, darauf hin,
daI. es der
Mangel an Nahrungsstoffen ini Blute ist, der die Hunger-
empfindung auslöst. p
g
Die Vermutung, es rnöehte die Stelle dos Zeiitralnerven-
systems, welche durch
den Mangel an Nahrungstoffen im
Blute gereizt wird, nahe dem Atmungszentrum und nahe dein
Uebergang des Gehirns in das verlängerte Mark gelegen sein,
kimen wir nirn durch
verschiedene Tatsachen begründen. S
i
i
d ß E k
k
d
i l
P
i g
g
Es ist wohl anzunehmen, daß die Zentren für alle die
vegetativen Funktionen wie für die Regulation der Körper-
w à r m e durch die Vasomotoren und durch die Schweißdrüsen,
für die Regelung der Aufnahme des Sauerstoffes durch
die Atmung und der Aufnahme der Flüssigkeit und der
festen Nahrung nahe beisammen liegen, haben sie doch
vielfach Wechselbeziehungen zueinander. Sie müssen in dem
entwicklungagesehichtlich ältesten Teile des Gehirns, im Palae-
en e ep h al on ,
lokalisiert sein, sind doch diese vegetativen
Funktionen bei allen Tieren, ob hoch oder tief stehend, gleich-
mäßig ausgebildet. Mit unserem bewu ßten Handeln und
unserem hewn lIten Fühlen und gar mit der Intelligenz haben
sie ja wenig zu tun. Deshalb ist es auch von vornherein aus-
zuschließen, daß das nervöse Zentrum, von welchem die Auf-
nabme der flüssigen und der festen Nahrung reguliert wird,
im Großhirn zu suchen ist. So wissen wir, daß Erkrankungen der mittleren Partien
der Gehirnbasis, wie Basisfraktur oder Geschwülste der ilypo-
physe oder luetische Prozesse am Infundibulum, den ,,Hungei
nact Fliissigkeit", d. h. den Durst, stark steigern können, so-
daß es zum Diabetes insipidus kommt. Das Aufnahmebedürfnis
nach konsistenter Nahrung, der Hunger, ist nun bekanntlich
durchaus nicht immer von dem nach Flüssigkeit, vom Durste,
zu trennen. So hat der Siiugling nur Bedürfnis nach Aufnahme
Von Flüssigkeit. ll
il
i
i d ß
h
i
b So wissen wir, daß Erkrankungen der mittleren Partien
der Gehirnbasis, wie Basisfraktur oder Geschwülste der ilypo-
physe oder luetische Prozesse am Infundibulum, den ,,Hungei
nact Fliissigkeit", d. h. den Durst, stark steigern können, so-
daß es zum Diabetes insipidus kommt. Das Aufnahmebedürfnis
nach konsistenter Nahrung, der Hunger, ist nun bekanntlich
durchaus nicht immer von dem nach Flüssigkeit, vom Durste,
zu trennen. So hat der Siiugling nur Bedürfnis nach Aufnahme
Von Flüssigkeit. 5) Arbeiten aus der Psychiatrischen Klinik in Würzburg. Jena
1914 8. S. 690.
2) Würzburg 1913
3) zitiert nach A. Sohühler; Die Erkrankungen der Zirbeldiüse.
Handbuch der Neurologie, von Lewandowsky 4. ') Pflug. Arch. 51.
2) V h
dl
d Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. durcit die
Muskelempfindungen, welche von diesen ausgehen, wird
auch das Gro ßhirn von dem Bestehen eines Mangels an
Nahrungsatoffen unterrichtet,
hn1ieh wie uns erst die erhöhte
Atemfrequenz und ein Druck auf der Brust von dem Mangel
an Sauerstoff im Blute Kenntnis geben. d
H
hl Daneben verursacht aber der Hunger - wohl
y o rn
Zwisohenhirn aus - ein Unlustgefühl, das, wie jede
Art der Stimmung, schwer zu beschreiben ist, das aber schon
den Säugling zu lebhaften Aeußerungen der Unlust veranlaßt
und das den großhirnlosen Hund unruhig werden läßt. Daß
die Leerkontraktionen des Magens es sind, welche so an-
haltendes Schreien beim hungernden Säugling
verursachen,
ist nicht wahrscheinlich, beim älteren Individuum wenigstens
verursachen solche doch niemals wirklich schmerzhafte Emp-
findungen. Auch schreit der Säugling noch weiter, wenn er,
bevor er gesättigt, ,,gestilt" ist, von der Brust wieder ab-
gesetzt wird, obgleich nun der Magen nicht mehr leer ist. l d E
äh
d Durch
angestrengte geistige Tätigkeit,
durch
lebhafte Stimmungen (z. B. durch Angst und Kummer
oder durch Zorn) kann sowohl der Hunger wie die Geschlechts-
lust für einige Zeit verdrängt werden. Anderseits sind äußere
Eindrücke, wie der Anblick oder der Geruch von leckeren
Speisen, imstande, den Hunger auszulösen. Sie tun dies aber
nur dann, wenn das Gehirn unter dem Einfluß ungenügender
Nahrung steht. Verursachen ja auch sinnliche Eindrücke
nur in dem durch die inneren Sekrete der Geschlechtsdrüsen
erotisierten Gehirne die Gesohlechtslust. g
g
g
Sch]ießlich
führt
die mangelnde Ernährung des
Großhirns selbst auch zu gewissen Störungen. Das
Nachlassen der geistigen Spanukraft, die Arbeitsunlust, das
Oefühl der Schwäche und des Schwindels, das Flimmern, das
Ohrensausen und endlich die Ohnmacht, alle diese Erscheinungen
sind zweifellos auf Nachlaß der Nahrungszufuhr zu den Gro B -
hiruganglienzellen zurückzuführen. Sch]ießlich
führt
die mangelnde Ernährung des
Großhirns selbst auch zu gewissen Störungen. Das
Nachlassen der geistigen Spanukraft, die Arbeitsunlust, das
Oefühl der Schwäche und des Schwindels, das Flimmern, das
Ohrensausen und endlich die Ohnmacht, alle diese Erscheinungen
sind zweifellos auf Nachlaß der Nahrungszufuhr zu den Gro B -
hiruganglienzellen zurückzuführen. ist das Blut mit Nahrungsstoffen gesättigt, so werden
Speisen, auch wenn sie noch so ,,appetitlich" zubereitet sind,
keinen Appetit auslösen. Im Gegenteil ! Versucht man nun
trotzdem Nahrung zuzuführen, so wird man auf Widerwillen
stoßen, ja man wird Ekelgefiihi erzeugen. Der Widerwille
gegen weitere Nahrungsaufnahme besteht aber nicht nur bei
vollem Magen. Ekelerregende Eindrücke sind imstande, auch
bei leerem Magen jede Eßlust sofort zu unterdrücken. Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Wissen wir doch auch das Atem-
zentrum und den Ort, von dem die Gefäße und die Schweiß-
drüsen innerviert werden, nicht genau zu lokalisieren. Wahr-
scheinlich handelt es sich garnicht um scharf umschriebene
Zentren. Kurz, wir sind noch weit entfernt, uns über die
zentrale, d. h. zerebrale Beeinflussung der vegetativen Funk-
tionen eine richtige Vorstellung machen zu können. Daß es nicht die Hypophyse, daß vielmehr das Zwischen-
hirn es ist, von welchem das Hunger- und Durstgefühl aus-
geht, das ist aus den Beobachtungen zu entnehmen, welche
auch bei den seltenen Tumoren der Glandula pinealis eine
vermehrte und abnorme Eßlust feststellten. Nach Kurz,
Oestreich und Slawyk, Daly und Neumann3) ist die
Polyphagie eines der Symptome der Erkrankungen der Zirbel-
drüse; sie wird auf die Reizung zurückgeführt, welche diese
Tumoren an den Wandungen des dritten Ventrikels verursachen. Besonders interessant war mir eine Mitteilung von Geheim-
rat Nissi (Heidelberg), daß in den Wandungen des dritten
Ventrikels sich Ganglienzellen sympathischen Charakters finden,
Zellen, die denen des Nucleus visceralis vagi oder denen des
Seitenhornes im Rückenmark völlig gleichen. Mit Bestimmtheit glauben wir aber doch annehmen zu
dürfen, daß im Zentralnervensystem eine Stelle ist, deren
Ganglienzellen durch den Mangel an rasch abbaufähigen Stoffen
im Blute gereizt werden, und daß diese Reizung dann über
den Vagus Leerkontraktionen des Magens und Saftsekretion
dort auslöst. h
b
i
l
i h g g
Alle die körperlichen Erscheinungen, die mit der Hunger-
empfindung einhergehen, werden vom Zwischenhirn und von
den nahegelegenen Teilen der Medulla oblongata ausgelöst und
innerviert; so liegen im Anfangsteile des verlängerten Markes
die Kerne für den Vagus (Nucleus visceralis vagi am Boden
des vierten Ventrikels), und von dort aus werden die Magen. Nach unserer Ueberzeugung ist es also nicht
die Leere des Magens, sondern der Mangel des Blutes
an Nahrungsstoffen, welcher das Gurren und das
Druckgefiihl in der Magengegend verursacht. Das
Druckgefühl ist durch die Muskelspannungen des leeren Magens
bedingt. Diese werden von uris ebenso empfunden, wie auch
die Muskelspannungen des Enddarmes, welche die Defäkation 173* 173* Nr.44 DXtTSCHE M
IZII,USCKK WOOHENSOEfl1}. '1Oo gelöst wird. ( l)iabetes insipidus bei Lilsionen des Infundi. bulums.) einleiten, empfunden werden. Auch im Enddarm können
Leerkontraktionen als Tenesmus recht störend werden. U b i
h
h
E FI W b
')
di Mi
d )
Die Unterscheidung zwischen dem Begriff ,,Jlunger" und
dem Begriff ,,Appetit" wird nicht scharf durchzuführen sein. ') In Wagners Handwörterbucb der Physiologie III. Teil p. 580,
zitiert nach C. y. Voit: Abschnitt Hunger in Herr manna Handbuch
der Physiologie dea Menschen 6.
2) K
li Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Leichtere Grade des Hungers, aber insbesondere das Be-
dürfnis nach einer bestimmten Art der Nahrung,
nennen wir Appetit. Uebrigens hat schon E. FI. Weber ') um die Mitte des
vorigen Jahrhunderts die Vermutung ausgesprochen. daß es
sich beim Hunger um Muskelgefühle handle, die durch die
Zusammenziehungen des leeren Magens verursacht wethen. g
g
Uebor diese Leerkontraktionen des Magens sind in den
letzten Sahren von amerikanischen Autoren, so von Cannon
und Waahburn und von Canson, eine Reihe von Arbeiten
veröffentlicht worden, die sich zum Teil auf Selbstversuche
gründen. Diese Porseher konnten durch Gummiblasen, die
sie mit einer Schlucksonde in den Magen einführten, fest-
stèllen, daß sieh jedesmal mit dem Auftreten von Hunger
empfindungen Kontraktionen des Magens einstellten und daß
sich diese Kontraktionen nicht ur auf den Magen beschriinkten,
sondern daß sie auch auf den Endteil des Oesophagus und
den Anfangeteil des Darmes übergingen. pp
Nach einer reichlichen Mahlzeit und nach Stilung des
Hungers kann noch Appetit auf eine Tasse Kaffee bestehen. D
A
i
h l
i h
H
h li h
i di g
pp
Der Appetit verhält sich zum Hunger ähnlich, wie die
auf eine Persönlichkeit beschränkte Liebe zu dem rohen Go-
schlechtetriebe, dem es nur auf die Betätigung des Geschlechts-
alites ankommt. Appetit und Liebe sind die auf ein bestimmtes
Objekt gerichteten Aeußerungen des Hungers und der Ge-
sehlechtslust. Dieses Dokument wurde zum persönlichen Gebrauch heruntergeladen. Vervielfältigung nur mit Zustimmung des Verlages. Aehnlich wie die inneren Sekretionsprodukte, welche die
Geschlechtsdrüsen in das Blut abgeben, das Gehirn ,,eroti-
sieren" und so die Sucht nach Geschlechtabetätigung aus-
lösen, verursacht der Mangel an rasch abbaufähigen Stoffen
im Gehirn die Sucht nach Nahrungsaufnahme. Und ähnlich
wie die im Gehirn entstehende Oeschlechtslust zu gewissen
körperlichen Veränderungen,
so zur Erectio membri oder
Zur Sekretion der Intröitusdrüsen führt, verursacht der im
Gehirn zustandekommende Hunger von dort aus, auch über
das vegetative Nervensystem, Sekretion dÑ Speichel- und
Magendrüsen und peristaltische Kontraktionen des
leeren
Magens. i i
T i k i
d
h Durch die Leerkontraktionen des Magens bzw. durcit die
Muskelempfindungen, welche von diesen ausgehen, wird
auch das Gro ßhirn von dem Bestehen eines Mangels an
Nahrungsatoffen unterrichtet,
hn1ieh wie uns erst die erhöhte
Atemfrequenz und ein Druck auf der Brust von dem Mangel
an Sauerstoff im Blute Kenntnis geben. d
H
hl Durch die Leerkontraktionen des Magens bzw. Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Der
Hunger wird eben nicht vorn Magen, sondern vom Gehirn
ausgelöst. g
g
Charakteristisch für den Hunger sind diese Empfindungen,
wie das Schwindel- und Schwitchegefühl, nicht. Das Großhirn
spricht ja auch auf andere Schäden, wie auf Ermüdung oder
auf Blutarmut, selbst wenn die Nahrungezufuhr genügend ist. mit Schwäche und Schwindel an. üb
di Eingehende psychologische Studien
über die
Beein.. flussung geistiger Leistungen durch Hungern" verdanken wir
W. Weigandt ). Wenn die mangelnde geistige Leistungs-
fähigkeit von dem hungernden Individuum selbst erkannt wird,
dann trägt sie ja auch indirekt zur Hungerempfindung bei. Der Hunger kann sich auf be stirn mt e Nahrungestoffe
beschränken. Das Wild, welches nur von Pflanzenstoffen lebt,
wandert weit, um salzhaltigere Nahrung zu finden. Auch wir
Menschen haben bei einseitiger Ernährung, selbst wenn wir
uns an ihr satt essen können, Hunger auf die Stoffe, die uns
gerade fehlen. Beim Mittagsmahl kann es uns unmöglich sein,
von einer F'leischkost noch mehr zu essen, während wir das
Bedürfnis haben, Kohlehydrate vielleicht in Form einer süßen
Speise oder von Kompott noch in größeren Mengen aufzu-
nehmen. Diese unsere Ueberzeugung wird schließlich auch durch
die Erfolge biw. durch die Mißerfolge unserer Therapie be-
kräftigt. Nie noch ist es uns gelungen, einen Mangel an Appetit
durch Medikamente, die auf den Magen einwirken, wie durch
Arnara oder durch Salzsäureverabreichung oder durch so viele
gepriesene Mittel wie durch das Tropon, das Sanatogen oder
die Somatose zu beheben. Wohl aber ist es möglich, durch
Einwirkung auf das Gehirn, wie durch appetitreizende Z. bereitung der Speisen, durch den Geruch und den Geschmack
einer Fleischbrühe - ,,l'appetit vient en mangant" - die
daniederliegende Eßlust wieder zu erwecken. Das beste Mittel
zur Erzeugung des Appetits scheint uns freilich möglichst lange
Nahrungsenthaltung zu sein. Erst dann, wenn alle resorbier-
baren Stoffe des Blutes vom Körper aufgesaugt und verbraucht
sind, wird der Mangel an Nahrung im Blute vom Zentral-
nervensystem aus diejenigen Erscheinungen auslösen, die uns
den Hunger empfinden lassen. Eingehende psychologische Studien
über die
Beein.. flussung geistiger Leistungen durch Hungern" verdanken wir
W. Weigandt ). Wenn die mangelnde geistige Leistungs-
fähigkeit von dem hungernden Individuum selbst erkannt wird,
dann trägt sie ja auch indirekt zur Hungerempfindung bei. Der Hunger kann sich auf be stirn mt e Nahrungestoffe
beschränken. Das Wild, welches nur von Pflanzenstoffen lebt,
wandert weit, um salzhaltigere Nahrung zu finden. er Physiologie dea Menschen 6.
2) Kraepelin
Psychologische Arbeiten 4. Aus der Medizinischen Poliklinik der Universitht in Würzburg.
Ueber die Hungerempfindung.)
Von Prof. Dr. L. R. Müller,
Oberstabsarzt und Chefarzt des Reservelazaretts ,,}Tauger Schule" in
Würzburg. Auch wir
Menschen haben bei einseitiger Ernährung, selbst wenn wir
uns an ihr satt essen können, Hunger auf die Stoffe, die uns
gerade fehlen. Beim Mittagsmahl kann es uns unmöglich sein,
von einer F'leischkost noch mehr zu essen, während wir das
Bedürfnis haben, Kohlehydrate vielleicht in Form einer süßen
Speise oder von Kompott noch in größeren Mengen aufzu-
nehmen. Wir dürfen doch wohl kaum den Magenwänden die Fähig-
keit zutiauen, beurteilen zu können, was unserem Körper zu
seinem Bestande oder zu seinem Aufbau noch fehlt. Und so
scheint: unS auchi die Tatsache, daß sich der Hunger auf be-
stimmte Stoffe beshränken kann, auf seine zentrale Aus-
lösung hinzuweisen. Jedenfalls dürfen wir annehmen, daß das
Bedürfnis zu einer genossenen Nahrung noch Flüssigkeit auf-
zunehmen, von den basalen Teilen des Zwischenhirns aus- Freilich werden pathologische Vorgänge, wie der
Uebergang von Eirischmelzungs- und Abbauprodukten des
eigenen Körpereiweißes ins Blut bei der Kachexie oder bei
Infektionskrankheiten oder wie die starke Beeinträchti-
gung der wärmeregulatorisehen Zentren im Zwischenhirn beim
Fieber, ein Bedürfnis nach Nahrungsaufnahme nicht
auf-
kommen lassen. So kommt es, daß gerade die Störungen, welche
mit starkem Kräfteverlust einhergehen, oft zu einer so hart-
näckigen Appetitlosigkeit, ja zu einem Widerwillen gegen
Fleisch führen. 1301 DEUTSCHE MEDIZiNISCHE WOOITENSOHRIFT. 28. Oktober 1915 Mit unserer Auffassung von der zentralen Auslösung der
Hungerempfinthtng läßt sich der Mangel des Appetits, wie er
mit manchen Nagenerkrankungen, insbesondere mit den Magen-
,,verstimmungefl" einhergeht, schwer erklären. k
i h
ll
d ß d
d
h ll Man könnte sich nur vorstellen, daß der durch allzu-
reichliche oder durch
ungeeignete Nahrung
, verstimmte"
Magen nicht oder nui ungenügend auf die Reize reagiert,
welche vom Zwischenhirn über den Vague zu seinen Drüsen
und zu seiner Muskulatur gelangen, und daß deshalb die Hunger-
kontraktionen und damit die örtlichen Hungerempfindungen
nicht zustandekommen. Jedenfalls gehen manche Formen
der Appetitlosigkeit mit der Empfindung des Aufgetriebenseins
oder des Voilseins des Magens, also mit einer mangelhaften
Kontraktion der Magenmuskulatur einher. Der Kontraktions-
zustand des Magens spielt sicherlich bei der Entstehung des
Hungers eine Rolle. g
Vielleicht kommt es aber auch in dem erkrankten Magen-
T)armkanal, wie z. B. bei der akuten Gastroenteritis, zur Bil-
dung von Stoffen, von Hormonen oder von Giften, welche die
vegetativen Anteile des Zwischenhirns beeinflussen und so
eine Hungerempfindung nicht aufkommen lassen. g
p
g
Wenn im höheren Alter die Lebhaftigkeit der Hunger-
empfindungen nachläßt, so müssen wir eben bedenken, daß
das Bedürfnis nach Nahrungsaufnahme geradeso wie das Be-
dürfnis nach Fortpflanzung ein Ausdruck der Vitalität ist. Mit dem Nachlaß der Lebenskraft, mit dem Alter leiden beide
Triebe. Die Stilung des Hungers geht mit Nachlaß der Unlust-
gefühle, ja mit ausgesprochenen Lustempfindungen einher. So sorgt die Natur für die Erhaltung des Individuums. Daß
die Stillung des Hungers dem Menschen einen bewu ßten
Genuß verschafft, das ist ein Vorzug, den er vor dem Tiere hat. S hl ß i)
hi
b
D l Schluß. i)en hier gegebenen Darlegungen mag entnommen
werden, daß das Hungergefühl keine einheitliche Emp-
findung ist. Es setzt sich vielmehr aus mehreren Organ-
empfindungen zusammen. Die Vorgänge, die diesen Organ-
empfindungen zugrundeliegen, wie der Speichelfiuß, die Hunger-
kontraktionen des Magens scheinen vom Palaeeneephalon aus-
gelöst zu werden. Die Verarmung des Blutes an abbaufähigen
Stoffen ist es wohl, die diese Innervationen verursacht. Aber
auch im Neencephalon im Großhirn bedingt der Mangel des
Blutes an Nährstoffen gewisse Organempfindungen, die sich
in
Beeinträchtigung
der
geistigen
Leistungsfähigkeit,
im
Schwindel oder im Flimmern vor den Augen und in Schwäche-
zuständen äußern können.
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https://openalex.org/W3016185218
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Adding pieces to the Alice in wonderland syndrome puzzle: a comment to the paper by Brooks and colleagues
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Arquivos de Neuro-Psiquiatria
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Adding pieces to the Alice in wonderland
syndrome puzzle: a comment to the
paper by Brooks and colleagues
Adicionando peças ao quebra-cabeça da síndrome de Alice no país das maravilhas: in the development of AIWS symptoms. As presented by
Brooks and colleagues, most lesions in the reported case
series are located very close to this region. Even in our case,
AIWS was followed by migrainous headache. Symptoms’ tim-
ing was consistent with an aura rather than a seizure (more-
over, EEG excluded epileptic activity), suggesting that AIWS
was associated to the patient’s first migraine aura. Dear Editor, We have read with great interest the paper by Brooks
and colleagues about their patient experiencing Alice in
Wonderland syndrome (AIWS), following a brain hemor-
rhage. Interestingly, the patient had a past medical history of
migraine with aura (MA); and AIWS, although secondary to a
hemorrhage, was followed by a pulsatile headache1. We appre-
ciate the effort of the authors to combine results from differ-
ent case reports to draw a clearer pattern of AIWS. To date,
the pathophysiology of AIWS remains elusive and far to be
fully understood. By reading the paper by Brooks et al.1, two
questions come to mind. First, is AIWS due to alterations in a
specific brain region or rather a more complex fronto-tempo-
ral-parietal network? Second, why are patients with migraine
more prone to experience AIWS than the other neurological
patients, and why do they often have migraine at the same
time they have AIWS? g
In this line, a second case3, of a 47-year-old woman, who
suffered from MA may help clarifying how AIWS and MA are
related. The patient had visual MA since her adolescence. Few years ago, she started having a progressive modifica-
tion of her aura features. Instead of her typical scotoma, she
experienced a mosaic vision, that was subsequently associ-
ated to feelings of elongation and dismemberment of her left
arm, depersonalization and slowing in temporal perception. These episodes lasted up to six hours and were followed by
migrainous headache. She started a pharmacological ther-
apy for major depression, and, after some lines of treatment,
she was prescribed with aripiprazole. She discontinued and
restarted aripiprazole several times, according to her mood
and, invariantly, on the first day of treatment with aripiprazole
she experienced the AIWS symptoms previously described. We performed a SPECT during an episode of AIWS and found
a marked hypoperfusion of the right primary somatosensory
area with a corresponding hyperperfusion of the homolateral
precuneus. 1IRCCS Fondazione Don Carlo Gnocchi, Milano, Italia.
2University of Lausanne, Centre Hospitalier Universitaire Vaudois (CHUV), My Space Lab, Department of Clinical Neuroscience, Lausanne, Switzerland.
3University of Geneva School of Medicine, Developmental Imaging and Psychopathology Laboratory, Geneva, Switzerland.
4Sapienza Università di Roma, Dipartimento di Neuroscienze Umane, Roma, Italia.
5Consorzio Universitario per i Disordini Adattativi e la Cefalea, Pavia, Italia.
PaviaAlessandro VIGANÒ
https://orcid.org/0000-0002-8079-5354; Giulio MAESTRIA
https://orcid.org/0000-0003-2900-1480;
Valentina MANCINI
https://orcid.org/0000-0003-4411-896X; Vittorio DI PIERO
https://orcid.org/0000-0002-2631-7562
Correspondence: Alessandro Viganò; Fundação IRCCS Don Carlo Gnocchi, Via A. Capecelatro 66, 20148, MIlan, Italia; E-mail: alessandro.vigano1@unimi.it
Conflict of interest: There is no conflict of interest to declare.
Received on November 5, 2019; Accepted on November 15, 2019. https://doi.org/10.1590/0004-282X20190194 https://doi.org/10.1590/0004-282X20190194 LETTER 1.
Brooks JBB, Prosdocimi FC, Rosa PBD, Fragoso YD. Alice in
Wonderland syndrome: "Who in the world am I?". Arq Neuropsiquiatr.
2019 Sep 23;77(9):672-4. http://dx.doi.org/10.1590/0004-
282x20190094 Adding pieces to the Alice in wonderland
syndrome puzzle: a comment to the
paper by Brooks and colleagues
Adicionando peças ao quebra-cabeça da síndrome de Alice no país das maravilhas: The hypoperfusion area was compatible with that
involved in the cortical spreading depression (CSD), the neu-
ral correlate of the aura4. Below, we reported two cases that might help in finding
answers to these questions. Recently, we described the occur-
rence of AIWS associated to migraine headache in a 54-year-
old man, with a history of migraine without aura (MoA)2. The patient did not have migraine attacks in the year prior to
the AIWS. During the AIWS episode, he saw computer icons
going out of the screen and moving in the space between him
and the screen. Neuroimaging investigations showed a glio-
blastoma on the left temporal-occipital junction (TOJ). This
case suggested that TOJ and associative areas of the parietal-
temporal-occipital carrefour are probably the most involved 242 the volume of inputs inflow and the inner representation of
the self. Associative area with a higher level of multimodal-
ity (e.g. TOJ, cuneous/precuneous) can more easily induce
intense alterations of the representation between the self
and enviroment5. Interestingly, the case reported by Brooks
and colleagues showed a lesion in the precuneus area. In our review on the topic5, we hypothesized that one
of the main sources of AIWS symptoms is an imbalance
between primary and secondary sensory regions. In our
hypothesis, AIWS can be triggered by an anatomical or func-
tional alteration of the normal information flow through sen-
sory networks. Migraineurs are more likely to develop CSD,
which is an example of a transient and fully reversible func-
tional impairment of the neuronal activity, occurring mainly
in cortical primary areas. CSD might, therefore, facilitate the
disconnection of these primary areas from their respective
secondary associative areas, producing a mismatch between In conclusion, although there is encouraging evidence
that we will reach a comprehensive insight on the pathophys-
iology of AIWS, further investigations on neuronal correlates
of AIWS are needed to obtain a valid theory. 2.
Mastria G, Mancini V, Viganò A, De Sanctis R, Letteri F, Toscano
M, et al. Temporal-occipital glioblastoma presenting with Alice
in Wonderland Syndrome in a patient with a long-time history of
migraine without aura. Neurocase. 2018 Oct/Dec;24(5-6):242-4.
https://doi.org/10.1080/13554794.2018.1562079 3.
Mancini V, Mastria G, Frantellizzi V, Viganò A, Petsas N, Sollaku S, et
al. Aripiprazole-Triggered Alice in Wonderland Syndrome Episodes
Studied with 99mTc-HMPAO Brain SPECT. Eur Neurol. 2018;79(5-
6):333-4. https://doi.org/10.1159/000490902
4.
Charles AC, Baca SM. Cortical spreading depression and migraine. Nat
Rev Neurol. 2013;9(11):637-44. https://doi.org/10.1038/nrneurol.2013.192
5.
Mastria G, Mancini V, Viganò A, Di Piero V. Alice in Wonderland
Syndrome: a clinical and pathophysiological review. Biomed Res Int.
2016;2016:8243145. https://doi.org/10.1155/2016/8243145 References 1. Brooks JBB, Prosdocimi FC, Rosa PBD, Fragoso YD. Alice in
Wonderland syndrome: "Who in the world am I?". Arq Neuropsiquiatr. 2019 Sep 23;77(9):672-4. http://dx.doi.org/10.1590/0004-
282x20190094
2. Mastria G, Mancini V, Viganò A, De Sanctis R, Letteri F, Toscano
M, et al. Temporal-occipital glioblastoma presenting with Alice
in Wonderland Syndrome in a patient with a long-time history of
migraine without aura. Neurocase. 2018 Oct/Dec;24(5-6):242-4. https://doi.org/10.1080/13554794.2018.1562079
3. Mancini V, Mastria G, Frantellizzi V, Viganò A, Petsas N, Sollaku S, et
al. Aripiprazole-Triggered Alice in Wonderland Syndrome Episodes
Studied with 99mTc-HMPAO Brain SPECT. Eur Neurol. 2018;79(5-
6):333-4. https://doi.org/10.1159/000490902
4. Charles AC, Baca SM. Cortical spreading depression and migraine. Nat
Rev Neurol. 2013;9(11):637-44. https://doi.org/10.1038/nrneurol.2013.192
5. Mastria G, Mancini V, Viganò A, Di Piero V. Alice in Wonderland
Syndrome: a clinical and pathophysiological review. Biomed Res Int. 2016;2016:8243145. https://doi.org/10.1155/2016/8243145 243 Viganò A et al. The pathophysiology of AIWS
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https://rshare.library.torontomu.ca/articles/journal_contribution/A_CMOS_Smart_Temperature_and_Humidity_Sensor_with_Combined_Readout/21534555/1/files/38171214.pdf
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English
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A CMOS Smart Temperature and Humidity Sensor with Combined Readout
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cc-by
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Sensors 2014, 14, 17192-17211; doi:10.3390/s140917192 Sensors 2014, 14, 17192-17211; doi:10.3390/s140917192 sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
OPEN ACCESS sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
OPEN ACCESS Keywords: capacitive sensor; humidity measurement; microelectronic implants; oscillator;
on-chip sensor; temperature sensor 1. Introduction Temperature and humidity sensors are widely used in many measurement and control applications,
including process control, meteorology, agriculture, battery-powered systems and medical equipment [1–8]. Temperature is a major concern in active implanted medical devices, especially in situations where
neural stimulators are located in close proximity to the neural tissue [9]. To protect patients from harm
due to the heat dissipated from implantable stimulators, the ISO 14708-3 [10] requires that no outer
surface of an implantable part be greater than 2 °C above the normal surrounding body temperature,
either in normal operation or single-fault condition. Any higher temperature rises can only be justified
if the manufacturer convincingly demonstrates its safety for a particular application [10]. An on-chip
temperature sensor calibrated for a small temperature range (e.g., 35 °C to 40 °C), but which is very
sensitive to small changes (e.g., 0.1 °C) will ensure the safe operation of a stimulator located on the
same chip. The requirements for sensors used for on-chip thermal management of non-implantable
processors are different due to their allowable wider temperature range and the stability of the supply
voltage [11,12]. While it has been demonstrated that a high level of absolute accuracy can be achieved
with a complementary metal-oxide semiconductor (CMOS) smart temperature sensor [13], relative
changes (from the initial value on implantation) are more important for implant monitoring, and they
require a high level of sensitivity. Moisture is another major concern for electronic devices operated in humid environments or
implanted in the body. Moisture that has penetrated the device package eventually causes condensation
on the active area of the integrated circuit, leading to corrosion, performance deterioration and device
failure. The most direct way to check that the micro-package [14] is functional and remains dry is to
measure its internal relative humidity (RH). A humidity sensor using a 0.6 μm CMOS process with
on-chip readout electronics is reported in [15]. An interdigitated capacitor that is covered by an
inorganic passivation layer followed by a polyimide overcoat forms the moisture sensitive sensor. It does not require any post-processing steps. However, the area required for the sensor itself is
relatively large (4 mm2 in [15]). It is desirable to provide a small sensor that can be added, for
example, to micro-packaged implantable stimulator chips [9,16] without a large increase in total area. Clemens Eder 1, Virgilio Valente 1, Nick Donaldson 2 and Andreas Demosthenous 1,* Keywords: capacitive sensor; humidity measurement; microelectronic implants; oscillator;
on-chip sensor; temperature sensor Sensors 2014, 14 Sensors 2014, 14 17193 Clemens Eder 1, Virgilio Valente 1, Nick Donaldson 2 and Andreas Demosthenous 1,* Clemens Eder 1, Virgilio Valente 1, Nick Donaldson 2 and Andreas Demosthenou 1 Department of Electronic and Electrical Engineering, University College London, Torrington P
London WC1E 7JE, UK; E-Mails: c.eder@ucl.ac.uk (C.E.); v.valente@ucl.ac.uk (V.V.) 1 Department of Electronic and Electrical Engineering, University College London, Torrington Place,
London WC1E 7JE, UK; E-Mails: c.eder@ucl.ac.uk (C.E.); v.valente@ucl.ac.uk (V.V.)
2 Department of Medical Physics and Bioengineering, University College London, Malet Place,
WC1E 6BT, UK; E-Mail: n.donaldson@ucl.ac.uk London WC1E 7JE, UK; E-Mails: c.eder@ucl.ac.uk (C.E.); v.valente@ucl.ac.uk (V.V.)
2 Department of Medical Physics and Bioengineering, University College London, Malet Place,
WC1E 6BT, UK; E-Mail: n.donaldson@ucl.ac.uk 2 Department of Medical Physics and Bioengineering, University College London, Malet Place,
WC1E 6BT, UK; E-Mail: n.donaldson@ucl.ac.uk * Author to whom correspondence should be addressed; E-Mail: a.demosthenous@ucl.ac.uk;
Tel.: +44-20-7679-3189; Fax: +44-20-7388-9325. * Author to whom correspondence should be addressed; E-Mail: a.demosthenous@ucl.ac.uk;
Tel.: +44-20-7679-3189; Fax: +44-20-7388-9325. Received: 15 July 2014; in revised form: 25 August 2014 / Accepted: 3 September 2014 /
Published: 16 September 2014 Received: 15 July 2014; in revised form: 25 August 2014 / Accepted: 3 September 2014 /
Published: 16 September 2014 Abstract: A fully-integrated complementary metal-oxide semiconductor (CMOS) sensor
for combined temperature and humidity measurements is presented. The main purpose of
the device is to monitor the hermeticity of micro-packages for implanted integrated circuits
and to ensure their safe operation by monitoring the operating temperature and humidity
on-chip. The smart sensor has two modes of operation, in which either the temperature or
humidity is converted into a digital code representing a frequency ratio between two
oscillators. This ratio is determined by the ratios of the timing capacitances and bias
currents in both oscillators. The reference oscillator is biased by a current whose
temperature dependency is complementary to the proportional to absolute temperature
(PTAT) current. For the temperature measurement, this results in an exceptional
normalized sensitivity of about 0.77%/°C at the accepted expense of reduced linearity. The
humidity sensor is a capacitor, whose value varies linearly with relative humidity (RH)
with a normalized sensitivity of 0.055%/% RH. For comparison, two versions of the
humidity sensor with an area of either 0.2 mm2 or 1.2 mm2 were fabricated in a commercial
0.18 μm CMOS process. The on-chip readout electronics operate from a 5 V power supply
and consume a current of approximately 85 µA. 2. Architectural Overview Figure 1 shows the architecture of the combined temperature and humidity sensor. The readout is
based on the ratiometric frequency measurement of two relaxation oscillators. Two modes of operation
are selectable by the T/RH control signal, either the temperature mode (TMOD) or the relative
humidity mode (RHMOD). The operation of the reference oscillator (REF-OSC) is the same in both
modes. A bandgap circuit (BG) generates temperature-independent voltage levels of 1 V and 2 V,
which are used to set the thresholds of the window comparators in the oscillator. REF-OSC is always
connected to the same reference capacitor (CREF) and supplied by bias current IREF, biasing its internal
current source/sink. This current is generated in BG and has a negative temperature coefficient;
therefore, the frequency of REF-OSC decreases with an increase in temperature. Figure 1. Architecture of the combined temperature and humidity sensor. Depending on
the measurement mode, one of the counters (CNT1 or CNT2) defines the reference period
for the other counter. BG, bandgap circuit; REF-OSC, reference oscillator; T/RH,
temperature/relative humidity; PTAT, proportional to absolute temperature; PISO,
parallel-in/serial-out; DTR, data ready; SCLK, serial clock. In the TMOD of operation, REF-OSC and one 16-bit counter (CNT1) generate a reference time
interval. Any temperature increase leads to an increase in the proportional to absolute temperature
(PTAT) current, increasing the frequency of the readout oscillator (TRH-OSC) and the rate at which
the readout counter CNT2 counts. When CNT1 overflows, the instantaneous value of CNT2 is
sampled. Note that the frequencies of REF-OSC and TRH-OSC are moving in opposite direction with
increasing temperature, which improves the sensitivity of the temperature measurement. The
consequences for linearity will be elaborated in Section 3. In the RHMOD of operation, the frequency of TRH-OSC decreases with increasing capacitance,
which is directly proportional to RH (i.e., C = f(RH)). In this mode, the roles of CNT1 and CNT2 are In the TMOD of operation, REF-OSC and one 16-bit counter (CNT1) generate a reference time
interval. Any temperature increase leads to an increase in the proportional to absolute temperature
(PTAT) current, increasing the frequency of the readout oscillator (TRH-OSC) and the rate at which
the readout counter CNT2 counts. When CNT1 overflows, the instantaneous value of CNT2 is
sampled. Note that the frequencies of REF-OSC and TRH-OSC are moving in opposite direction with
increasing temperature, which improves the sensitivity of the temperature measurement. 1. Introduction This paper presents a combined temperature and RH sensor with a common readout, which
simplifies the design and significantly reduces the chip area needed. The readout is based on
ratiometric counter values in which a measurement counter is driven by a relaxation oscillator. Its
frequency depends on either a temperature-dependent current charging a constant capacitor or on a
constant current charging a humidity-dependent capacitor. By associating the reference counter to a
current with opposite temperature coefficients, the temperature sensitivity can be increased (as will be
shown). The combination of small area, supply voltage independence, high sensitivity to both
temperature and humidity changes, as well as the ease of readout makes the design concept very
attractive for active implantable epidural electrodes incorporating several stimulator chips [16]. For
monitoring purposes in active implantable microsystems, it is the relative changes in temperature and
humidity (and not their absolute values) that are important in order to trigger an alarm. The paper is organized as follows. Section 2 describes the concept overview, and Section 3 provides
a sensitivity analysis for both humidity and temperature. Section 4 presents details of the circuit Sensors 2014, 14 Sensors 2014, 14 17194 design. The measured results follow in Section 5. Finally, the discussion and concluding remarks are
presented in Sections 6 and 7. 3. Sensitivity Analysis The timing of the oscillator waveform is shown in Figure 2. During one half period (tON), the
capacitor C is charged from the comparator reference voltage level V1 to V2 and: The timing of the oscillator waveform is shown in Figure 2. During one half period (tON), the
capacitor C is charged from the comparator reference voltage level V1 to V2 and: τ
τ
τ
= +
=
Δ
=
−
=
ON
2
1
1
(
)
( )
t t
t
V
V
V
I t d
C
(1) (1) Figure 2. Triangular waveform on the capacitor and output waveform as a function of
time. The duty cycle is assumed to be 50%. For a bias current ( )
I t
I
=
and a 50% duty cycle:
ON
OSC
2
1
1
2 (
)
I
V
I t
f
C
C V
V
Δ
=
⋅
⋅
=
−
(2)
where fOSC is the oscillator frequency. The frequency is directly proportional to current and inversely
roportional to capacitance. Consequently, the roles of the reference and measurement counters must
e interchanged when switching from one mode to another CNT1 is the reference counter for TMOD For a bias current ( )
I t
I
=
and a 50% duty cycle:
1
I For a bias current ( )
I t
I
=
and a 50% duty cycle: ON
OSC
2
1
1
2 (
)
I
V
I t
f
C
C V
V
Δ
=
⋅
⋅
=
−
(2) ON
OSC
2
1
1
2 (
)
I
V
I t
f
C
C V
V
Δ
=
⋅
⋅
=
−
(2) (2) where fOSC is the oscillator frequency. The frequency is directly proportional to current and inversely
proportional to capacitance. Consequently, the roles of the reference and measurement counters must
be interchanged when switching from one mode to another. CNT1 is the reference counter for TMOD,
while CNT2 is for RHMOD. The two modes of operation are therefore treated separately below. idity Sensor Mode (RHMOD) 2. Architectural Overview The
consequences for linearity will be elaborated in Section 3. In the RHMOD of operation, the frequency of TRH-OSC decreases with increasing capacitance,
which is directly proportional to RH (i.e., C = f(RH)). In this mode, the roles of CNT1 and CNT2 are
therefore interchanged, where CNT2 defines the measurement interval and CNT1 is engaged for
readout. Note that TRH-OSC is biased by the same current as REF-OSC; thus, to a first order Sensors 2014, 14 17195 Sensors 2014, 14 approximation, the temperature dependence of the current is cancelled. The frequency measurement
is only affected by changes of the sensor capacitance, which ideally depends on humidity only. The following analysis examines the performance of both modes of operation in terms of sensitivity
and linearity. 3.1.1. Humidity Dependency Equation (5) is independent of the current ratio and only dependent on the capacitance ratio, that is: Equation (5) is independent of the current ratio and only dependent on the capacitance ratio, that is: =
⋅
±
=
⋅
±
16
16
REF
RH
CNT1
RH
REF
(RH)
2
0.5
2
0.5
f
C
N
f
C
(6) (6) Since CREF can be considered constant and does not depend on RH, the change in counter value
with humidity is: Since CREF can be considered constant and does not depend on RH, the change in counter value
with humidity is: ∂
∂
=
⋅
⋅
=
⋅
⋅
∂
∂
16
16
CNT1
RH
RH
REF
REF
(RH)
(RH)
1
2
NSRH 2
RH
RH
N
C
C
C
C
(7) (7) where NSRH is the normalized sensitivity to RH, which is of the order of 0.073%/% RH [15]. For
equal capacitances, the counter change is of the order of 730 ppm/RH·216 ≈ 48, and the least significant
bit (LSB) resolution is therefore 1/48 ≈ 0.02% RH. Sensors 2014, 14 Sensors 2014, 14 17196 where fREF is the REF-OSC frequency. The uncertainty of half a count, i.e., the quantization error, is
taken into account. Equation (4) shows that the reference counter value is a measurement of the
frequency ratio of the oscillators. As the frequencies are dependent on both current and capacitance
(see Equation (2)), the frequency ratio may be expressed as the ratiometric measurement of either
currents or capacitances: =
⋅
±
=
⋅
⋅
±
16
16
REF
REF
RH
CNT1
RH
REF
REF
( )
( ,RH)
2
0.5
2
0.5
( )
( )
f
I
T
C
T
N
f
C
T
I
T
(5) (5) where CREF is the reference capacitor, CRH is the humidity sensitive capacitor, IREF is the REF-OSC
current and T is the temperature. It is assumed that neither the capacitance of the reference capacitor
nor the amplitude of the current is affected by RH. For equal bias currents, the sampled counter value
NCNT1 is therefore only dependent on the capacitive ratio CRH/CREF. Two cases are considered. 3.1. Humidity Sensor Mode (RHMOD) The measurement interval, TRH, is determined by the TRH-OSC frequency, fRH, and the bit size of
the overflow counter, CNT2. For a 16-bit counter, the measurement interval is: The measurement interval, TRH, is determined by the TRH-OSC frequency, fRH, and the bit size of
the overflow counter, CNT2. For a 16-bit counter, the measurement interval is: =
=
16
CNT2
RH
RH
RH
2
N
T
f
f
(3) (3) where NCNT2 is the counter value of CNT2. During this interval, the reference counter counts up to a
value NREF, which depends both on the frequency of the measured signal and the measurement
interval. Hence, =
⋅
=
⋅
±
16
REF
CNT1
REF
RH
RH
2
0 5
f
N
f
T
. f
(4) (4) 3.1.2. Temperature Dependency Here, Equation (6) is modified to take the temperature dependency of the reference capacitance
into account: =
⋅
±
=
⋅
±
16
16
REF
RH
CNT1
RH
REF
( )
2
0.5
2
0.5
( )
f
C
T
N
f
C
T
(8) (8) e change in the counter value with temperature is: The change in the counter value with temperature is: The change in the counter value with temperature is: The change in the counter value with temperature is: (
)
∂
∂
∂
=
⋅
−
⋅
∂
∂
∂
16
CNT1
RH
REF
REF
RH
2
REF
( )
( )
1
( )
( )
2
( )
N
C
T
C
T
C
T
C
T
T
T
T
C
T
(9) (9) This dependency only cancels in the ideal case when both capacitances and their temperature
coefficients are equal. For any other case, the temperature coefficient of a capacitor, TTC, is given by: This dependency only cancels in the ideal case when both capacitances and their temperature
coefficients are equal. For any other case, the temperature coefficient of a capacitor, TTC, is given by: Sensors 2014, 14 Sensors 2014, 14 17197 ∂
=
⋅
∂
0
1
( )
(
)
C T
TCC
C T
T
(10) (10) where C(T0) is the capacitance at temperature T0. A typical value of TCC for a poly-poly capacitor
CREF in a typical 0.18 µm CMOS process is 20 ppm/°C. Metal-dielectric-metal capacitors have higher
temperature coefficients, and the simulated temperature coefficient of the top-metal humidity sensor is
about 60 ppm/°C. e expressed in terms of TTC as: Equation (7) can be expressed in terms of TTC as: (
)
∂
=
−
⋅
∂
16
CNT1
RH
RH
REF
REF
( )
2
( )
N
C
T
TCC
TCC
T
C
T
(11) (11) where TCCRH and TCCREF are, respectively, the temperature coefficient of capacitor CRH and CREF. For
equal capacitances, the change in the counter value is about 40 ppm/°C · 216 = 2.62/°C. A temperature
increase of about 18 °C appears as a 1% RH increase. This temperature sensitivity is acceptable in
implants where temperature variations are low. 3.2. Temperature Sensor Mode (TMOD) 3.2.1. Temperature Dependency TCR can be very small for a poly-resistor
(−40 ppm/°C), which is much smaller than the TCIT at room temperature (about 3300 ppm/°C). 3.2.2. TCIREF < 0, Temperature Dependency F < 0, Temperature Dependency The derivative of a current ratio can be expressed in a similar manner to Equation (11) as: The derivative of a current ratio can be expressed in a similar manner to Equation (11) as: (
)
∂
=
⋅
−
=
⋅
−
−
∂
T
T
T
T
REF
REF
REF
REF
REF
1
I
I
I
TCI
TCI
TCR
TCI
T
I
I
I
T
(17) (17) The dependency of the relative value of CNT2 depends on temperature and is: The dependency of the relative value of CNT2 depends on temperature and is: The dependency of the relative value of CNT2 depends on temperature and is:
(
)
(
)
∂
=
⋅
⋅
−
+
⋅
∂
16
CNT2
T
REF
REF
REF
T
1 1
2
N
I
C
T TCI
TCR
T
I
C
T
(18) (
)
(
)
∂
=
⋅
⋅
−
+
⋅
∂
16
CNT2
T
REF
REF
REF
T
1 1
2
N
I
C
T TCI
TCR
T
I
C
T
(18) (18) Since the temperature coefficient of the bandgap was simulated close to zero and the temperature
coefficient of the n-well resistor used in BG is TCR = 3000 ppm/°C, the reference current will
necessarily have a negative temperature coefficient of TCIREF = −3000 ppm/°C. Together with the
chosen poly-type resistor for the PTAT current generation (−1400 ppm/°C), the sum of both
coefficients is −4400 ppm/°C, which is subtracted from the 3300 ppm/°C of the PTAT current at 300 K. This presents a boost in sensitivity by a factor of 2.3 with respect to a PTAT current generator. The
change in the counter value is 7700 ppm/°C · 216 ≈ 505, corresponding to an LSB resolution of
1/505 ≈ 0.002 °C. However, the increase in sensitivity is at the expense of increased non-linearity. The
analysis of the latter is not trivial, and a qualitative illustration of the trade-off between linearity and
sensitivity is shown instead in Figure 3. 3.2.1. Temperature Dependency The temperature sensitivity of the counter stage is first examined for a reference current IREF with
zero temperature coefficient (i.e., TCIREF = 0). The roles of measurement and reference counters are
now reversed (Figure 1). Equation (5) assumes the form: =
⋅
±
=
⋅
⋅
±
16
16
T
T
REF
CNT2
REF
REF
T
( ,RH)
( )
2
0.5
2
0.5
( )
f
I
T
C
T
N
f
I
C
T
(12) (12) where fT is the TRH-OSC frequency (in TMOD), and the capacitors CT and CREF are of the same type
with equal temperature coefficients. Thus, the derivative of the quotient (which can be derived in a
similar manner to Equations (8) to (11)) is: (
)
∂
=
−
=
∂
REF
REF
REF
T
T
T
( )
( )
0
( )
( )
C
T
C
T
TCC
TCC
T
C
T
C
T
(13) (13) The temperature, therefore, depends only on the PTAT current (IT) multiplied by the scaling
factor CREF/CT: The temperature, therefore, depends only on the PTAT current (IT) multiplied by the scaling
factor CREF/CT:
∂
∂
=
⋅
∂
∂
16
CNT2
REF
T
REF
T
( )
1
. 2
N
C
I
T
T
I
C
T
(14) (14) where IT is assumed independent of RH. The temperature coefficient of the current is given by [17]: ∂
∂
∂
=
⋅
=
⋅
−
⋅
=
−
∂
∂
∂
T
EB12
T
T
EB12
1
1
1
1
I
V
R
TCI
TCR
I
T
V
T
R
T
T
(15) (15) where VEB12 is the voltage difference between the differently biased bipolar transistors (Figure 5a) and
TCR denotes the temperature coefficient of a resistor. Therefore, Equation (14) can be written as: 17198 Sensors 2014, 14 17198 (
)
∂
=
−
⋅
⋅
∂
16
CNT2
T
REF
REF
T
1
. 1
2
N
I
C
T TCR
T
I
C
T
(16) (16) The term T·TCR introduces a non-linearity. TCR can be very small for a poly-resistor
(−40 ppm/°C), which is much smaller than the TCIT at room temperature (about 3300 ppm/°C). 3.2.2. TCIREF < 0, Temperature Dependency The term T·TCR introduces a non-linearity. 3.2.1. Temperature Dependency As the sensor will be used for temperature monitoring in an
implant where the temperature range of interest is restricted, the errors due to non-linearity are
small (Figure 3). Figure 3. Qualitative illustration of the compromise between linearity and sensitivity of the
ratio between PTAT and reference currents. (a) A reference current with a low negative
temperature coefficient will result in increased overall sensor sensitivity while minimally
compromising linearity; (b) Sensitivity can be further increased with a reference current of
a higher negative temperature coefficient, at the cost of increased deviation from linearity. (a)
(b) ( ) (b) (a) 17199 Sensors 2014, 14 Sensors 2014, 14 4.2.1. Bandgap Circuit Both the temperature-independent voltage references V1 and V2, and a temperature-dependent
reference current IREF are generated (Figure 5a). The topology is based on summation of a PTAT
current and a complementary-to-absolute temperature (CTAT) current, which is a commonly used
design in short channel processes [17]. A self-biased cascode current source (M8–M15) is used to hold
Nodes A and B at the same voltage, VEB (base-emitter voltage) of Q1. The cascode current mirror
requires about 2.3 V of compliance. The PTAT current is generated via the VEB1–VEB2 difference over
R1 and added to the CTAT current generated by VEB1 over R2 and R3. The sum of the currents is
mirrored to R4 and R5, which generate temperature-independent voltage drops of 1 V (V1) and 2 V (V2). The temperature dependence of V1 or V2 is a function only of the ratio of the values of R2 and R1 and
the number (N = 8) of elements used in Q2 [17]. All resistors were selected as the n-well type, because
of the positive temperature coefficient of about 3000 ppm/°C. This yields a negative TCIREF, as
previously explained. 4.2.2. PTAT Circuit The PTAT current source is shown in Figure 5b, and its topology is similar to the bandgap
reference (without the CTAT current). The poly-resistor has a negative temperature coefficient of
−1400 ppm/K, in order to further increase the temperature sensitivity of the output current. The startup
circuit is identical to the one used in the bandgap reference. 4.1. Humidity Sensor The capacitive humidity sensor was constructed as in [15,18] and requires no post-processing
steps. It is based on a capacitor consisting of an interdigitated finger structure formed by the top metal
layer with its inorganic passivation coating. The structure is covered by a moisture sensitive film,
which is formed by the readily available polyimide overcoat. A cross-section of the fabricated sensor
in [15] is shown in Figure 4. In the proposed design, the fingers are 3 µm wide and spaced 2.5 µm apart (the minimum width and
spacing allowed by the design rules of the technology; 0.18 µm X-FAB XP018). Two types of sensor
were implemented with sensing capacitors of different sizes. A 1 mm × 1 mm (15 pF simulated
capacitance between fingers) and a 300 µm × 300 µm (1.1 pF simulated capacitance) were fabricated
to investigate the sensitivity in these small structures. The larger sensor was accessible via pads to test
its capacitance as a function of humidity. The chip also contained circuit structures enabling testing of
individual blocks, such as the bandgap (BG) and PTAT reference outputs. Figure 4. Cross-section for three of the sensing capacitor’s fingers in [15] sectioned by
focused ion beam. Figure 5. (a) Reference current and voltage source. All resistors are of the n-well type;
(b) PTAT current source. e 5. (a) Reference current and voltage source. All resistors are of the n-well type; Figure 5. (a) Reference current and voltage source. All resistors are of the n-well type;
(b) PTAT current source. (a)
(b) (a) (a) (b) 17200 Sensors 2014, 14 Sensors 2014, 14 4.2. Bandgap and PTAT Designs Sensors 2014, 14 Sensors 2014, 14 Sensors 2014, 14 and U3 and are in the ns range. The undershoot that occurs before the flip-flop toggles causes
negligible timing errors (Figure 6b). Figure 6. (a) Relaxation oscillator with a single comparator; (b) oscillator timing diagram
(dimensions are not to scale). Figure 6. (a) Relaxation oscillator with a single comparator; (b) oscillator timing diagram
(dimensions are not to scale). g
( )
g
p
; ( )
g
g
(dimensions are not to scale). (a)
(b)
4.4. Control Logic
The control logic was designed in Verilog and consists of a simple state machine. A start signal first
sets CAPset to high, while enabling the bandgap reference (Figure 6a). It resets the output of the D-flip
flop, which connects the gate of M10 to M7. However, the current mirror M2–M3 is not enabled, thus
the drain of M7 is at VDD (5 V), turning off M7. The node at VCAP is therefore high impedance, and the
timing capacitor charges up to V1 via the switch M10. The time constant is defined by the capacitance
and the output resistance of the circuit generating V1, which is R4||R5 and is about 2.7 µs for the larger
humidity capacitor. A pulse of 20 µs is used to safely pre-charge the capacitor before turning off
CAPset and starting the triangular oscillation of VCAP from the lower threshold V1. Each oscillator
output is connected to a separate counter, whose frequency is recorded in a parallel-in/serial-out (a)
(b)
4.4. Control Logic
The control logic was designed in Verilog and consists of a simple state machine. A (b) 4.3. Relaxation Oscillator The relaxation oscillator is shown in Figure 6a. Prior to the start of the oscillation, the timing
capacitor C is connected to the high impedance node formed by the inactive output transistors of the
current source (M10) and sink (M6). The capacitor set (CAPset) signal resets the output and connects
the output node to the lower threshold voltage. The capacitor will be rapidly charged to 1 V, allowing
even the very first charging interval tON (Figure 2) to be accurately defined. The oscillator starts by
driving CAPset to “0” and the oscillator enable (EN) signal to “1”, enabling the bias current through
the 1:1 current mirrors formed by M2, M3, M6 and M7, M10. Output transistors M10 and M6 are
alternately switched off (by M5, M9) or connected to the current mirror through M4, M8. This design uses a single comparator implemented as an uncompensated two-stage op-amp, saving
the power consumption of a second comparator. The relaxation oscillator is based on charging and
discharging the timing capacitor between two well-defined voltages. The threshold values are switched
depending on whether the capacitor is being charged or discharged. This is accomplished by the XOR
gate U2 and the D-flip flop U3. When the capacitor voltage reaches the lower bound, the output of the
comparator is set to the positive supply rail (Figure 6b). The inverted output of U3 is still at 0 V,
causing U2 to be set to “1”, which resets U3 and causes the output of U2 to change back to “0”. The
duration for which the output of U2 stays high is mainly determined by the propagation delays of U2 17201 4.4. Control Logic The control logic was designed in Verilog and consists of a simple state machine. A start signal first
sets CAPset to high, while enabling the bandgap reference (Figure 6a). It resets the output of the D-flip
flop, which connects the gate of M10 to M7. However, the current mirror M2–M3 is not enabled, thus
the drain of M7 is at VDD (5 V), turning off M7. The node at VCAP is therefore high impedance, and the
timing capacitor charges up to V1 via the switch M10. The time constant is defined by the capacitance
and the output resistance of the circuit generating V1, which is R4||R5 and is about 2.7 µs for the larger
humidity capacitor. A pulse of 20 µs is used to safely pre-charge the capacitor before turning off
CAPset and starting the triangular oscillation of VCAP from the lower threshold V1. Each oscillator
output is connected to a separate counter, whose frequency is recorded in a parallel-in/serial-out The control logic was designed in Verilog and consists of a simple state machine. A start signal first
sets CAPset to high, while enabling the bandgap reference (Figure 6a). It resets the output of the D-flip
flop, which connects the gate of M10 to M7. However, the current mirror M2–M3 is not enabled, thus
the drain of M7 is at VDD (5 V), turning off M7. The node at VCAP is therefore high impedance, and the
timing capacitor charges up to V1 via the switch M10. The time constant is defined by the capacitance
and the output resistance of the circuit generating V1, which is R4||R5 and is about 2.7 µs for the larger
humidity capacitor. A pulse of 20 µs is used to safely pre-charge the capacitor before turning off
CAPset and starting the triangular oscillation of VCAP from the lower threshold V1. Each oscillator
output is connected to a separate counter, whose frequency is recorded in a parallel-in/serial-out Sensors 2014, 14 Sensors 2014, 14 17202 (PISO) register by the overflow of the counter (their roles are determined according to the T/RH mode
signal in Figure 1). Once the data transfer occurs, a data ready (DTR) signal is set high to indicate the
end of the measurement. The recorded value is then ready to be clocked out from the PISO by the
external serial clock (SCLK) (Figure 1). 5. Measured Results Two versions of prototype chips were fabricated in a 0.18-μm CMOS process (X-FAB XP018). Microphotographs of the chips are shown in Figure 7 (Version 1 and Version 2 chips). The larger
1 mm × 1 mm capacitor (humidity sensor) is visible on the top layer in Figure 7a. The readout circuit
occupies an area of 350 µm × 580 µm. The different test-structures of the readout (Circuits B1 to B6 in
Figure 7a) were first used to characterize the temperature and humidity sensitivity of the individual
blocks. These test structures are only available in the Version 1 chip, and they represent functional
blocks of the readout (which is nearly identical in both versions). The only difference in the readout in
the Version 2 chip is that the control logic part is omitted. This design choice had to be made as the
number of available pads was limited, and it was justified by confirming the correct operation of the
control logic in the Version 1 chip. For the sensor characterization, an MKF 240 environmental simulation chamber was used
(BINDER GmbH, Tuttlingen, Germany). All measurements were performed with the test printed
circuit board inside the chamber, connected by cables through access ports. Capacitance measurements
were performed with a Wayne Kerr 6500B precision impedance analyser (Wayne Kerr Electronics
Inc., Woburn, MA, USA). Short bursts at the output of both oscillators were sampled at 1 MHz using a
NI-USB-6353 acquisition card (National Instruments, Austin, TX, USA). A frequency counter
(Agilent 53131A, Santa Clara, CA, USA) was used for the direct measurement of the frequency ratio
between the outputs of the two oscillators. A summary of the overall performance is given in Table 1. Figure 7. (a) (A) Chip microphotograph of Version 1 chip (individual blocks are not
visible due to the top-metal dummy structure) and (B) layout with omitted top metal layer. Test structures: (B1) readout circuit, (B2) bandgap, (B3) PTAT, (B4) biasing stage,
(B5) relaxation oscillator, (B6) comparator. (b) Chip microphotograph of Version 2 chip
with smaller sensing capacitor: (I) readout circuit, (II) capacitive sensing element. (a)
(b) (b) (a) (b) 17203 Sensors 2014, 14 Table 1. Summary of performance. Table 1. Summary of performance. 5. Measured Results y
p
General
Technology
0.18 µm CMOS 5 M + THKMET
VDD
+5 V
IDD a
85 μA
Area b
0.2 mm2 (readout in both sensors)
Sensor Output
Temperature
Humidity
Sensitivity c
486/°C
124/% RH
Sensitivity error d
< ±4.3%
< ±14.5%
Temperature Sensor
Area
0.023 mm2 (part of readout circuit)
Sensitivity
7753 ppm/°C@37°C
Linearity
Compromise between linearity and sensitivity
(see text); ideal for TCIREF = 0. Capacitive Humidity Sensor
Version 1
Version 2
Area
1 mm2
0.09 mm2
Sensitivity e
514 ppm/% RH
584 ppm/% RH
a Static quiescent current; b readout only; c as change in the counter output per measurement unit;
d maximum variation for the batch (N = 14) of Version 1; e obtained by measurement of capacitive changes in
Version 1 and by frequency ratio in Version 2. 5.1. Capacitance vs. Relative Humidity (Version 1) General 5.1. Capacitance vs. Relative Humidity (Version 1) All 14 sensors of Version 1 showed a linear capacitance response as a function of humidity
(Figure 8), where the coefficient of determination is greater than 0.99. The mean response is: =
⋅
+
(pF)
0.0133 RH(%)
25.92 pF
C
(19) (19) where the 99% confidence interval (CI) is (25.8, 25.99) pF for the intercept and (0.0125, 0.0141) pF/%
for the slope. The normalized mean sensitivity is 0.0133/25.9 = 514 ppm/% RH at 0% RH. Figure 8. Measured capacitance versus relative humidity at 37 °C. Figure 8. Measured capacitance versus relative humidity at 37 °C. Figure 8. Measured capacitance versus relative humidity at 37 °C. Sensors 2014, 14 17204 Sensors 2014, 14 5.2. Temperature Dependency of Reference (Version 1) 5.2. Temperature Dependency of Reference (Version 1) The reference current followed a linear trend with a negative temperature coefficient (Figure 9a):
μ
= −
⋅
°
+
μ
REF( A)
0.00794
( C)
2.88
A
I
T
(20) (20) where the 99% CI is (2.81, 2.95) µA for the intercept and (−0.0082, −0.0077) µA/°C for the slope. The
average sensitivity is −0.00789/2.88 µA = − 2771 ppm/°C at 0 °C. The nominal simulated response
was practically identical to the measured average response, which had a slope <3000 ppm/°C. This
would be the slope expected from the quoted TCR of the n-well resistor of 3000 ppm/°C, if the
reference voltage was ideal and had a zero temperature coefficient. The measurement of the reference voltage V1 is shown in Figure 9b. Analysis of the results showed
a 99% CI for the intercept between 1 V and 1.04 V, with a mean slope of 322 µV/°C, corresponding to
a temperature coefficient of 316 ppm/°C. The variation of the gradients has a 99% CI of (248, 397) µV/°C. The sensitivity of V1 to temperature variations is attributed to the spread of the characteristics of the
n-well resistors. Figure 9. (a) Measured reference currents with temperature; (b) Measured temperature
dependency of all reference voltages V1. (a) (a)
(b)
5.3. Temperature Dependency of PTAT (Version 1) (b) (b) (a) 5.3. Temperature Dependency of PTAT (Version 1) The measured PTAT current varies with temperature as expected (Figure 10): The measured PTAT current varies with temperature as expected (Figure 10): μ
= −
⋅
°
+
μ
PTAT( A)
0.011
( C)
2.05 A
I
T
(21) μ
= −
⋅
°
+
μ
PTAT( A)
0.011
( C)
2.05 A
I
T
(21) (21) where the 99% CI is (1.995, 2.098) µA for the intercept and (10.6, 11.2) nA/°C for the slope. This
yields a normalized temperature coefficient of 0.011/2.05 = 5366 ppm/°C at 0 °C. 17205 Sensors 2014, 14 Sensors 2014, 14 Figure 10. Measured PTAT currents versus temperature. 5.4. Measured Frequency Output (Version 2) 5.4. Measured Frequency Output (Version 2)
The measured conversion from current to frequency (TMOD) over the frequency range 20–70 °C
produced oscillator output frequencies in the range of 90–108 kHz. Similarly, the measured conversion
from capacitance to frequency (RHMOD) over the humidity range 20%–80% RH produced oscillator
output frequencies in the range of 26.6–27.1 kHz. 5.2. Temperature Dependency of Reference (Version 1) There was an anomaly in the intermediate range, and
extensive post-layout simulations revealed that this was due to the shared bandgap reference (Figure 1)
and can be corrected by providing isolated bandgap references. The behaviour of the measured oscillator frequency ratio between 20% RH and 45% RH for a 5.4. Measured Frequency Output (Version 2) The measured conversion from current to frequency (TMOD) over the frequency range 20–70 °C
produced oscillator output frequencies in the range of 90–108 kHz. Similarly, the measured conversion
from capacitance to frequency (RHMOD) over the humidity range 20%–80% RH produced oscillator
output frequencies in the range of 26.6–27.1 kHz. There was an anomaly in the intermediate range, and
extensive post-layout simulations revealed that this was due to the shared bandgap reference (Figure 1)
and can be corrected by providing isolated bandgap references. The behaviour of the measured oscillator frequency ratio between 20% RH and 45% RH for a
representative chip from Version 2 is shown in Figure 11. The directly measured frequency ratio
fREF/fRH is shown. The ratio follows a linear trend with humidity, where the slopes are dependent on the
temperature (0.0016/% RH at 37 °C and 0.0023/% RH at 47 °C). The normalized sensitivity at 37 °C
and 0% RH is 909 ppm/% RH, where both intercepts nearly meet. The temperature error at 0% RH
would be 1% RH per 2 °C, which is lower than what was expected from Equation (11). The reasons
behind this temperature dependency are discussed in Section 6. Figure 11. Measurement of frequency ratios in Version 2. Figure 11. Measurement of frequency ratios in Version 2. Figure 11. Measurement of frequency ratios in Version 2. Sensors 2014, 14 17206 Figure 12 shows the oscillogram of the oscillator outputs in the temperature mode, where the
reference oscillator correctly stops after 65,535 cycles. The DTR signal is set and is automatically reset
after all of the data has been clocked out (not shown). Figure 12. Oscillogram of the oscillator outputs. TEMP, temperature signal; REF,
reference signal; DTR, data ready signal. 6. Discussion
DTR
REF
TEMP 6.2. Humidity Sensor The measured capacitance is about 10 pF higher than was expected from simulation. The reason for
this mismatch can be partly attributed to the parasitic capacitances. However, that may not be the only
reason why the sensitivity of 0.0514%/% RH is lower than the previously reported value of 0.077%/% RH
for a similar construction in a different CMOS process [15]. The lower sensitivity can be attributed to
the sum of minimum track width and spacing, which was 3 µm + 2.5 µm = 5.5 µm
(as defined by the design rules) in the 0.18-μm CMOS process. This is larger than the 5 µm in [15],
yielding a 10% lower density of electrodes per area. Furthermore, the gap distance of 2.5 µm was
lower than the computed optimum of 4 µm for a comparable electrode density [18]. Despite the reduced sensitivity of the sensing element, an LSB resolution of 1/124% RH is possible,
due to the 16-bit counter resolution. Repeated measurements of 100 consecutive readout values at a
humidity level of 20% RH showed a sample standard deviation of 65 counts; thus, a 3σ difference of
195 counts is detectable. A resolution of 2% RH is therefore achievable. It has been shown that for the
entire batch, an absolute accuracy of 6% could be achieved for a span of 40% RH. For smaller sensor
areas, the oscillator frequencies must increase, but this has the advantage of shorter conversion times. For Version 2 (300 μm × 300 μm sensor), the frequency was 1.8 MHz, and tens of MHz can be
achieved with CMOS relaxation oscillators. A higher sensitivity (909 ppm/% RH at 0% RH) was
found in that version, where the sensing capacitor was not accessible for capacitance measurement. This suggests that the sensitivity of the humidity measurement is not necessarily related to the total
area of the sensor when using the same shape of its outline. Measurements showed a temperature dependency (Figure 11) that is larger than expected (1% RH
per 2°C instead of 1% RH per 18 °C), but comparable to [15], where the output frequency changed
0.14% per 8 °C or 0.0175%/°C. This corresponds to a normalized sensitivity of the frequency output of
0.045%/°C for a 1% change per 2.6 °C. The output frequency as a function of humidity in [15] was not
only offset with higher temperatures, but its negative slope steepened, as well. 6.1. Temperature Sensor The temperature sensor was designed with two currents of opposing temperature coefficient. For the
reference current, a linear function of temperature was measured with a negative temperature
coefficient. The slope of the PTAT current was larger in measurements than in simulation (about
10%), which could be attributed to a 10% smaller resistor. This is within the tolerance of the
polysilicon resistor (R in Figure 5b) as quoted in the process specifications. However, the ratio
between CTAT and PTAT currents within the bandgap (BG) should only depend on the ratio between
resistors (which can be accurately matched) and on the collector current ratio between Q1 and Q2
(Figure 5a). In theory, the latter should not depend on the process spread [13]. However, the collector
current, Ic, depends on both the emitter current and a significant base current (as the current gain βF is
only 2.6 for the chosen process). Although the dependency of the current gain on the current is taken
into account in the provided Gummel–Poon model, the recombination effect for the base-emitter diode
presents a ‘knee’ in the base current versus voltage function and may not be accurately modelled [19]. Thus, the already small βF may significantly drop even further with smaller emitter-base voltage (VEB),
so the collector current tends to be even smaller for each of the eight bipolar junction transistors (BJTs)
in Q2, reducing the VEB of Q2 and, therefore, increasing VBE1–VBE2 and, consequently, the PTAT
current. Failure to accurately model the drop in βF = f(Ic) in the simulation could, therefore, explain the
discrepancies between simulation and results. An appropriate correction of the collector current ratio
would undoubtedly lead to better temperature stability of the reference voltage. Despite the suboptimal
performance, the temperature coefficient of the bandgap voltage (316 ppm/°C) is still more than an
order of magnitude lower than the temperature coefficient of the PTAT-to-reference current ratio. The Sensors 2014, 14 17207 oscillator frequency ratio is, therefore, mainly determined by the current, not the threshold voltages
(V1 and V2). The measured results confirmed the principle that the reference current can be designed to have a
negative temperature coefficient with a low spread of slopes. As the slopes are related to the
complementary temperature coefficient of the n-well resistors, it follows that their spread in the
temperature coefficient must be low, as well. 6.1. Temperature Sensor It was demonstrated that a compromise between linearity
and sensitivity can be achieved by choosing the type of resistors R1–R3 (Figure 5a) on the basis of a
desired temperature coefficient. Non-silicided P+ poly resistors, for example, can have a temperature
coefficient as low as 40 ppm/°C, which would greatly improve the linearity at the cost of
PTAT sensitivity. 6.3. Batch Calibration The data for the entire batch suggests that the temperature measurement error due to the variation of
the current slopes is small in comparison to the error due to the spread in the current intercept, that is,
the offset. For example, the range of the 99% CI (confidence interval) of the intercept is
2.098 µA – 1.995 µA = 103 nA for the PTAT current and 2.95 µA – 2.81 µA = 140 nA for the
reference current. However, in a temperature interval from 20 °C to 80 °C, the maximum error due to
the slope deviation would be much lower, that is (11.2 – 10.6) nA/°C · 60 °C = 36 nA for the PTAT
and (8.2 – 7.7) nA/°C · 60 °C = 30 nA for the reference current. It is, therefore, safe to say that for the
short temperature range of interest, a one-point calibration technique would be adequate for the batch
under consideration. This can be readily achieved by reading and storing the digital value at a defined
temperature and subtracting this value in subsequent measurements. The chip-to-chip variation in the RH measurement is dependent on the spread of the capacitive
sensing element. The 99% CI intercept range is 0.19 pF, whilst the error due to slope deviation
amounts to (0.0141 – 0.0125) pF/% · 100% = 0.16 pF for the entire measurement range. This suggests
that the slope of the sensor should be calibrated in those applications that require a high degree of
absolute accuracy, but the one-point calibration as described above would be sufficient to monitor
excessive moisture ingress in micro-packages with a compromised hermetic seal. The calibration of
the humidity sensor within the hermetically-sealed micro-package [14] could only be reliably
performed once it is ensured that the internal humidity is close to zero and that the properties of the
sensor are not affected by the bonding process. 6.2. Humidity Sensor This would mean that
the sensitivity would increase with increasing temperature. The reasons for this may lie in the
temperature dependence of the polyimide moisture absorption, which can be taken into consideration
by a temperature-dependent correction factor for the relative capacitance change with RH [20]. Interpolation of the experimentally obtained values showed that the factor varied from 1.04 at 37 °C to
1.086 at 47 °C, corresponding to an increase of 4.6% change. Although this is much smaller than 17208 Sensors 2014, 14 observed in the present paper or in [15], the temperature dependence of the humidity measurement is
unlikely to originate from temperature-dependent oscillator bias currents. The argument being that:
(i) the temperature coefficient of both reference currents should cancel each other out according to
Equation (8); (ii) if the temperature coefficient of the reference currents were dominant, then their
negative values would actually decrease the sensitivity with temperature; and (iii) the charge current
in [15] was temperature independent. Acknowledgments This work is part of the European Research project, NEUWalk (www.neuwalk.eu), funded by the
European Community’s Seventh Framework Programme (FP7/2007-2013) under Grant Agreement
No. 258654. 7. Conclusions A smart CMOS sensor with a combined readout for both temperature and humidity has been
presented. Circuits were designed to meet the needs of low current consumption, high sensitivity, no
post-processing and small size (see comparison in Table 2). These features were achieved using
common circuitry for both temperature and RH measurements. It was shown that the digital output of the sensors represents an indirect ratiometric measurement of
frequencies. A linear response was achieved for humidity-related capacitance changes with a
reasonable sensitivity. In this design, the implementation of a current source with a negative
temperature coefficient achieved a much higher sensitivity than a conventional PTAT, at the expense
of reduced linearity. The designer can find a compromise between sensitivity and linearity depending
on the requirement of the application. In this application, the aim was to reliably detect small
temperature changes in order to guarantee safe operation of an implantable chip and to set an alarm 17209 Sensors 2014, 14 when the integrity of the hermetically-sealed micro-package is compromised by detecting any ingress
of moisture. Table 2. Comparison of CMOS temperature/humidity sensors. Reference Type
Normalized RH
Sensitivity
(ppm/% RH)
Temperature
Resolution
(°C)
Current
Consumption
(μA)
Area
(mm2)
CMOS
Technology (μm)
[12]
T
n/a
0.05
10
0.12
0.35
[13]
T
n/a
0.03
25
4.5
0.7
[21]
T
n/a
0.25
20
0.18
0.18
[15]
RH
770
n/a
111
4.8
0.6
[22]
RH
1440
n/a
1
0.7
0.15
This work T/RH
584
7753 ppm/°C a
85
0.29
0.18
a In this work, it is the sensitivity that is important. Table 2. Comparison of CMOS temperature/humidity sensors. Future work is necessary to determine the temperature-dependent sensitivity of the employed
polyimide and how its sensitivity varies across an entire wafer. In addition, the high
temperatures involved in the micro-packaging process [14] may have an irreversible effect on the
short- and long-term vapour adsorption properties of the polyimide film, which should be
also investigated. Author Contributions Clemens Eder designed the chips, performed the experiments, analysed the data and drafted the
manuscript. Virgilio Valente helped with the circuit design. Nick Donaldson and Andreas Demosthenous
supervised the work and finalized the manuscript. Conflicts of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. References 1. Miloudi, M.; Remadnia, M.; Dragan, C.; Medles, K.; Tilmatine, A.; Dascalescu, L. Experimental
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No maternal or direct effects of ocean acidification on egg hatching in the Arctic copepod Calanus glacialis
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PloS one
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cc-by
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Editor: Erik V. Thuesen, Evergreen State College,
UNITED STATES Received: August 22, 2017
Accepted: January 24, 2018
Published: February 7, 2018 Copyright: © 2018 Thor et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. No maternal or direct effects of ocean
acidification on egg hatching in the Arctic
copepod Calanus glacialis Peter Thor1*, Fanny Vermandele2, Marie-Helene Carignan2, Sarah Jacque2, Piero Calosi2 Peter Thor1*, Fanny Vermandele2, Marie-Helene Carignan2, Sarah Jacque2, Piero Calosi2
1 Norwegian Polar Institute, Fram Centre, Tromsø, Norway, 2 Universite´ du Que´bec à Rimouski,
De´partement de Biologie Chimie et Ge´ographie, Rimouski, Canada ete
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1 Norwegian Polar Institute, Fram Centre, Tromsø, Norway, 2 Universite´ du Que´bec à Rimouski,
De´partement de Biologie Chimie et Ge´ographie, Rimouski, Canada 1 Norwegian Polar Institute, Fram Centre, Tromsø, Norway, 2 Universite´ du Que´bec à Rimouski,
De´partement de Biologie Chimie et Ge´ographie, Rimouski, Canada 1 Norwegian Polar Institute, Fram Centre, Tromsø, Norway, 2 Universite´ du Que´bec à Rimouski,
De´partement de Biologie Chimie et Ge´ographie, Rimouski, Canada * peter.thor@npolar.no * peter.thor@npolar.no a1111111111
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a1111111111 Abstract Widespread ocean acidification (OA) is transforming the chemistry of the global ocean and
the Arctic is recognised as the region where this transformation will occur at the fastest rate. Moreover, many Arctic species are considered less capable of tolerating OA due to their
lower capacity for acid-base regulation. This inability may put severe restraints on many fun-
damental functions, such as growth and reproductive investments, which ultimately may
result in reduced fitness. However, maternal effects may alleviate severe effects on the off-
spring rendering them more tolerant to OA. In a highly replicated experiment we studied
maternal and direct effects of OA predicted for the Arctic shelf seas on egg hatching time
and success in the keystone copepod species Calanus glacialis. We incubated females at
present day conditions (pHT 8.0) and year 2100 extreme conditions (pHT 7.5) during oogen-
esis and subsequently reciprocally transplanted laid eggs between these two conditions. Statistical tests showed no effects of maternal or direct exposure to OA at this level. We
hypothesise that C. glacialis may be physiologically adapted to egg production at low pH
since oogenesis can also take place at conditions of potentially low haemolymph pH of the
mother during hibernation in the deep. RESEARCH ARTICLE OPEN ACCESS Citation: Thor P, Vermandele F, Carignan M-H,
Jacque S, Calosi P (2018) No maternal or direct
effects of ocean acidification on egg hatching in the
Arctic copepod Calanus glacialis. PLoS ONE 13(2):
e0192496. https://doi.org/10.1371/journal. pone.0192496 Editor: Erik V. Thuesen, Evergreen State College,
UNITED STATES Ocean acidification effects in Calanus glacialis global riverine discharge. This discharge not only carries low H+ buffering capacity but also
contributes significant loads of terrestrial carbon, which increases CO2 production by promot-
ing heterotrophic microbial respiration [9]. Finally, inflow from the North Atlantic transports
increasing amounts of anthropogenic CO2 to the Arctic Ocean [10]. Arctic organisms are
therefore the first to face the effects of OA and will continue to experience stronger OA in the
future [7]. Competing interests: The authors have declared
that no competing interests exist. The magnitude of these changes extends beyond anything experienced during most species’
evolutionary history [11], and while significant effects are predicted for many marine animals
[12, 13], effects may be particularly conspicuous in the Arctic. Contrary to lower latitude eury-
thermal animals, true Polar species show low energetic costs for physiological maintenance at
low temperatures [14, 15]. This is, however, at the expense of a lower capacity for cellular
homeostasis and acid-base regulation [16]. The capacity to counter negative effects of OA is
therefore thought to be particularly low in Arctic species. Moreover, while direct effects from
the immediate environment will impose serious implications for sensitive organisms, effects
carried through the generations may convey either alleviation or additional stress. Adaptation
will enable increased tolerance to a changed environment [17], while maternal effects carried
to the offspring may act in either direction. Maternal effects can precondition offspring to the
environment experienced by mothers through cytoplasmic and mitochondrial factors pack-
aged into the egg during oogenesis [18–21]. However, under severe maternal stress these fac-
tors may be lacking or differently conditioned resulting in poorly developing offspring. For
instance, copepods mothers’ ingestion of low quality prey including harmful diatoms have
shown detrimental effects on egg hatching and subsequent naupliar development [22–24]. Such changes may have far-reaching consequences, and a depression of the long term year-to-
year recruitment of nauplii larvae or a delay in the timing of this recruitment can have signifi-
cant effects on the population development [25]. When these effects occur in ecologically
important species, they may spread beyond the species itself. Calanus glacialis constitutes a keystone species in the Arctic Ocean and adjacent seas [26–
28]. Introduction Uptake of anthropogenic CO2 is changing the chemistry of the global ocean [1]. When enter-
ing the sea, CO2 reacts with water to form carbonic acid, and this ocean acidification (OA) has
lowered the global ocean mean surface pH from 8.13 during the pre-industrial age to the pres-
ent day 8.05. This trend is predicted to continue and current models estimate a further
decrease of up to 0.4 pH units by the year 2100 [2–4]. In this respect, the Arctic is a region of
specific concern. OA is currently progressing at faster rates in many Arctic regions and is
expected to continue to do so [1, 5–7]. This is partly due to rising temperatures generating
increasing volumes of sea ice melt water, which holds low H+ buffering capacity [8]. Moreover,
while the Arctic Ocean contains only 1% of the global ocean volume, it receives 11% of the Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: The study was financially supported by a
grant from the FRAM High North Research Centre
for Climate and the Environment through the
Ocean Acidification and Ecosystem Effects in
Northern Waters Flagship to PT. The funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. 1 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 Preparation of incubation water For the initiation of incubations six 50 L buckets were filled with 0.3 μm filtered sea water
obtained from a seawater intake at 80 m depth (fsw). For the low pH treatment, small volumes
of fsw acidified to ca. pHNBS 5.5 (National Bureau of Standards scale) by CO2 bubbling (Map-
con CO2, Yara Praxair, Tromsø, Norway) were mixed into three of these buckets to reach a tar-
get pHNBS of 7.5 as measured by a Metrohm 826 pH meter equipped with a Metrohm LL
aquatrode electrode. Total scale pH (pHT) was determined spectrophotometrically in 12 mL
water samples pipetted in 5 cm quartz cuvettes. Absorbance was measured at 578 nm, 434 nm,
and 730 nm using a UV-2401PC spectrophotometer (UV-2401PC, Shimadzu Inc., Kyoto,
Japan) after addition of 10 μL of m-cresol-purple. pHT was then calculated according to Clay-
ton and Burne [43] with a dye-addition correction [44] and adjusted from measurement tem-
perature to in situ temperature according to Gieskes [45]. We did not measure a second
carbonate chemistry variable as in our previous OA studies [17, 32, 33, 46–49], and we were
unable to calculate the full carbonate chemistry, which is otherwise recommended [50]. For food, paste of the diatom Thalassiosira weissflogii (Tw 1200, Reed Mariculture, Camp-
bell, CA, USA) was added to a final concentration of ca. 10 μg Chl a L-1. Prior to the experi-
ment, the necessary dilution of the algal paste was established from the Chl a content of a 100x
dilution of the algal paste determined using a spectrophotometer (UV-2401PC, Shimadzu
Inc.) after overnight extraction in 70% ethanol [51]. Cell concentrations in this dilution was
established by cell counts under the microscope in a Bu¨rker-Tu¨rk counting chamber. The suit-
ability of the algal paste as prey for C. glacialis had been assured previously by comparing faecal
pellets counts from incubations of copepods with counts from copepods incubated at similar
concentrations of live T. weissflogii (Peter Thor, pers. comm.). Collection of copepods Calanus glacialis were caught by vertical tows, 100 m to the surface, with a 200 μm WP2 plank-
ton net (KC Research equipment, Silkeborg, Denmark) with a closed cod end in the Kongsfjord,
Svalbard (79.0˚ N, 11.7˚ E) in late May 2016. No specific authorisation was needed for collecting
copepods, and no endangered species were involved. On deck, the content of the cod end was
diluted in 25 L sea water produced from water collected at 80 m. Copepods were then trans-
ported to a cold room (5˚C) at the nearby Kings Bay Marine Laboratory (Ny-Ålesund, Sval-
bard). Calanus glacialis females were selected under the stereoscope using cut off plastic Pasteur
pipettes keeping all vessels on ice to avoid high temperatures. Individuals were identified to spe-
cies by number of pleopods and abdominal segments. They were distinguished from C. fin-
marchicus and C. hyperboreus on the basis of size [28], the presence of red pigmentation in the
antennules, a characteristic distinguishing C. glacialis from C. finmarchicus [42], and the lack of
lateral spikes on the distal prosome segment, which is a characteristic of C. hyperboreus. Several
hundred females were collected in a 50 L bucket to be distributed into incubation buckets. Along the continental shelf this species dominates in terms of biomass, may exert signifi-
cant grazing pressure on microplankton prey, and is a very important food item for many
pelagic predators such as carnivorous copepods and amphipods, but also Arctic fish species,
baleen whales, and marine birds [29–31]. Any changes to C. glacialis production will therefore
extend beyond the copepods themselves and potentially encompass a larger part of the entire
food web. As a consequence, much attention has been given C. glacialis and its possible future
in a changing Arctic [32–36]. In the present study, we investigated the effects of the immediate and maternal pH environ-
ment as predicted for the Arctic Ocean in year 2100 [1] on egg hatching dynamics in C. glacia-
lis. Egg hatching success (EHS) is not the only determinant, besides egg production rate, of
population recruitment success. Copepod eggs are often subjected to high mortality rates [37–
39], and while decreased EHS would have direct consequences for population recruitment, any
delay in the timing of egg hatching will increase individual predation risk. Moreover, delayed
hatching may increase the risk of phenological miss-match with the succession of the phyto-
plankton bloom rendering the developing larvae less adequately nourished [25]. We therefore
investigated the effects of OA on both EHS and egg hatching time. Differentiation between
maternal and direct effects was accomplished by reciprocally transplanting eggs produced by
females subjected to high and low pH. Calanus glacialis show both capital and income breed-
ing strategies [40, 41], so the study period was chosen late in spring to avoid contribution from
the winter energy capital and increase the possibility that all energy for egg production was
obtained from ingested food during the OA incubation. 2 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 Ocean acidification effects in Calanus glacialis Ocean acidification effects in Calanus glacialis After incubating for 7 d, all eggs were removed from the bottom of the buckets by siphon-
ing and the next day, all eggs produced from day 7 to day 8 were collected from each bucket,
also by siphoning. These eggs were collected on a 50 μm mesh size sieve, divided in two and
re-suspended in pre-prepared high and low pH incubation water in two 500 mL pitchers. From each of these pitchers, eggs were distributed by volume into nine replicate 50 mL flat cell
culture flasks (VWR Collection, Darmstadt, Germany) equipped with 50 μm mesh in the caps
to allow exchange with the outside water (total of 2 pH levels x 9 replicates x 6 buckets = 108
flasks). These flasks were then transplanted from the origin bucket to all six incubation buckets
according to the design in Fig 1 for the remainder of the experiment. The flask received on
average 21 ± 9 eggs (mean ± SD). Eggs were counted twice every day directly in the flasks
under a stereomicroscope until the fourth day after which they were counted once every day. Eggs where clearly visible through the flask walls. pHT was monitored as above once every day in all buckets, and when necessary, the water
was renewed by inserting a large 200 μm sieve into the water and siphoning off approximately
3/4 of the water in the bucket from inside of this sieve and replacing it with newly prepared
water. Algal concentration were measured by cell counts once every day and algae were added
to maintain target concentrations. Fig 1. Transplants of eggs among incubation buckets. For clarity only transplants in one treatment group (High to low pH; HL) are shown. The table shows all
transplants for each treatment group. Three flasks with eggs were transplanted along each of the arrows in the figure. In total, each treatment group contained nine
transplants. Fig 1. Transplants of eggs among incubation buckets. For clarity only transplants in one treatment group (High to low pH; HL) are shown. The table shows all
transplants for each treatment group. Three flasks with eggs were transplanted along each of the arrows in the figure. In total, each treatment group contained nine
transplants. https://doi org/10 1371/journal pone 0192496 g001 Fig 1. Transplants of eggs among incubation buckets. Copepod and egg incubations Collected C. glacialis females were released into 30 L of fsw, which was divided in six by vol-
ume. Copepods from these six batches were then caught on a 200 μm filter and re-introduced
immediately into the six incubation buckets. This started the 7 d incubation of the females. No
additional females were added during those 7 days. The water was kept in motion by gentle
bubbling and turned once every day to keep algal food in suspension. Ingestion was ascer-
tained by frequent observations of the copepods’ gut colour. 3 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 For clarity only transplants in one treatment group (High to low pH; HL) are shown. The table shows all
transplants for each treatment group. Three flasks with eggs were transplanted along each of the arrows in the figure. In total, each treatment group contained nine
transplants. Fig 1. Transplants of eggs among incubation buckets. For clarity only transplants in one treatment group (High to low pH; HL) are shown. The table shows all
transplants for each treatment group. Three flasks with eggs were transplanted along each of the arrows in the figure. In total, each treatment group contained nine
transplants. Fig 1. Transplants of eggs among incubation buckets. For clarity only transplants in one treatment group (High to low pH; HL) are shown. The table shows all
transplants for each treatment group. Three flasks with eggs were transplanted along each of the arrows in the figure. In total, each treatment group contained nine
transplants. https://doi.org/10.1371/journal.pone.0192496.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 4 / 12 Ocean acidification effects in Calanus glacialis Data treatment and statistical analyses For each flask, number of remaining eggs was converted to fraction hatched at every sampling
event. Egg hatching success (EHS) was calculated as the mean of fraction hatched from the last
three sampling events. To investigate possible delay effects of pHT in hatching, a sigmoid Hill
function was fitted to the cumulative increase in fraction hatched eggs over time in each flask. The Hill function outputs values of average time to hatching (Km) and maximum egg hatching
(E): ^e ¼
Eth
Kmhþth, where ê is predicted fraction hatched at time t and h is the sigmoid inflexion
factor. After removal of outliers (values further than two standard deviations from the me-
dian), values of EHS and Km (interpreted as average time to hatching) were compared among
the four treatment groups (high to high pH, high to low pH, low to high pH, and low to low
pH) by 1-factor permutational analysis of variance (PERMANOVA) on similarity matrices
assembled using Euclidian distances in Primer 6+ [52] employing a nested PERMANOVA
design: Treatment group + transplants(treatment group), where “transplants” were the specific
transplants among the treatment groups (i.e. the arrows in Fig 1). Average water temperature and pHT throughout the incubation period were compared
among incubation buckets by 1-factor PERMANOVA on similarity matrices assembled using
Euclidian distances. All PERMANOVA tests were followed by PERMDISP tests to verify the assumption of
homogeneity of multivariate dispersions. Ocean acidification effects in Calanus glacialis Fig 2. Cumulative egg hatching during the incubation period. To enable comparison among flasks holding different initial numbers of eggs,
egg counts were calculated as fractions of the number of eggs at the first count. Solid lines show mean predicted values from the Hill regressions
on each individual flask. The vertical lines at t = 2 d is inserted as a guide to facilitate visual judgement of differences among treatments. https://doi.org/10.1371/journal.pone.0192496.g002 Fig 2. Cumulative egg hatching during the incubation period. To enable comparison among flasks holding different initial numbers of eggs,
egg counts were calculated as fractions of the number of eggs at the first count. Solid lines show mean predicted values from the Hill regressions
on each individual flask. The vertical lines at t = 2 d is inserted as a guide to facilitate visual judgement of differences among treatments. https://doi.org/10.1371/journal.pone.0192496.g002 https://doi.org/10.1371/journal.pone.0192496.g002 https://doi.org/10.1371/journal.pone.0192496.g002 https://doi.org/10.1371/journal.pone.0192496.t002 Results There were no significant differences in average temperature among the incubation buckets
during the incubation period (Table 1; 1-factor PERMANOVA: Pseudo-F5,71 = 0.18, P = 0.98). pHT differed significantly between high and low pH treatments and there were no significant
difference among buckets within treatments (1-factor PERMANOVA pair-wise tests:
P > 0.05). There were no significant differences in diatom food concentration among buckets
(1-factor PERMANOVA: Pseud-F5,59 = 0.29, P = 0.91). In general egg hatching took place two days after collection of eggs from the bucket floors
(Fig 2). There were no significant differences in average time to hatching (Km) nor was there
any significant confounding effects from the nested transplant factor (1-factor PERMANOVA:
Treatment group: Pseudo-F3,93 = 0.70, P = 0.55, transplant(treatment group) Pseudo-F32,93 =
1.61, P = 0.057) (Table 2). Furthermore, there were no significant difference among the four
treatment groups in egg hatching success (EHS) (1-factor PERMANOVA: Treatment group:
Pseudo-F3,93 = 0.44, P = 0.72, transplant (treatment group) Pseudo-F32,93 = 0.99, P = 0.51)
(Table 2). Table 1. Averages of temperature (T), total scale pH (pHT), salinity (S), and algal concentration during the incubation period. Bucket
T
pHT
S
Algal concentration
˚C
μgChl a L-1
cells mL-1
High pH 1
5.48±0.22
8.01±0.05
35.12±0.07
10.00±2.92
8100±2371
High pH 2
5.45±0.31
7.96±0.09
35.12±0.04
9.48±1.72
7675±1402
High pH 3
5.51±0.30
7.97±0.05
35.12±0.04
8.74±4.54
7077±3688
Low pH 1
5.58±0.26
7.50±0.13
35.15±0.05
10.14±3.18
8222±2582
Low pH 2
5.52±0.32
7.48±0.15
35.16±0.05
10.10±3.28
8188±2663
Low pH 3
5.54±0.27
7.45±0.18
35.15±0.07
10.08±3.08
8162±2507
Means ± standard deviations. erages of temperature (T), total scale pH (pHT), salinity (S), and algal concentration during the incubation period. PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 5 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 Discussion A study of OA
effects on complete pelagic communities from the Baltic Sea revealed significant negative
effects of low pH (pH 7.45) on EHS [58, 59]. Interestingly, by transplanting eggs between low
and high pH environments, Vehmaa and colleagues showed that maternal effects can alleviate
pH effects on EHS but only at moderately reduced pH [58]. Eggs from females exposed to
moderately reduced pH experienced significantly higher EHS compared to eggs produced by
non-exposed females, contrary to what we observed in C. glacialis. Also, decreased pH may act
to change the egg hatching response to increasing water temperature. In Acartia sp., low pH
reduced the positive effect of increased temperature on EHS [60]. On the other hand, this
interacting effect was not seen in A. clausi where low pH reduced EHS at both high and low
temperature [55]. There is no calcification in the copepod exoskeleton and OA effects are car-
ried to copepods foremost by changes in hemolymph pH. Consequently, and because of logis-
tical constraints, we did not measure a second carbonate chemistry variable to enable
calculation of the full chemistry. Nevertheless, alkalinity is mostly stable and intermediate in
the Kongsfjord [61] and we expect no extremes of carbonate chemistry at the time of the
experiments. We studied egg hatching in C. glacialis despite the previous findings that EHS are not influ-
enced by pH levels similar to the present study [36] because EHS is not the only determinant
of population recruitment success. Copepods experience widely different predation risk
through their life cycle. Eggs and young nauplii larvae constitute important prey for pelagic
predators and consequently they experience high mortality rates [37–39]. Older nauplii and
copepodite stages possess more efficient behavioural escape mechanisms which lessen size
dependent predation risk [62, 63]. Thus, while decreased EHS would have direct consequences
for population recruitment, any temporal delay in the timing of egg hatching or development
in the young stages is equally detrimental since it increases individual life-time predation risk. Moreover, and perhaps more importantly, delayed hatching and development may increase
the risk of phenological miss-match with the succession of the phytoplankton bloom [25]. Although C. glacialis shows capital breeding in the ice-free Kongsfjord, they rely on the rela-
tively short ice algal and later pelagic phytoplankton blooms for the production of larvae in
most of the Arctic [41]. Discussion In the study presented here, we did not detect any significant direct or maternally transferred
effects of pH levels predicted for the Arctic Ocean in year 2100 on egg hatching success (EHS)
or egg hatching timing (Km) of Calanus glacialis eggs. Effects of pH on EHS has been studied previously in different Calanus species. In C. glacialis
egg hatching success was lower at pHNBS 6.9 [36], whereas pH levels more closely mimicking Table 2. Egg hatching success (EHS) and average time to hatching (Km) in the four treatments. Means ± standard
deviations. Values of Km are means of individual regressions on cumulative hatching from each incubation flask. Treatment
EHS
Km
d
HH
0.94 ± 0.06
2.02 ± 0.23
HL
0.92 ± 0.07
1.97 ± 0.16
LH
0.94 ± 0.05
2.05 ± 0.29
LL
0.89 ± 0.20
2.08 ± 0.24 Table 2. Egg hatching success (EHS) and average time to hatching (Km) in the four treatments. Means ± standard
deviations. Values of Km are means of individual regressions on cumulative hatching from each incubation flask. 6 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 Ocean acidification effects in Calanus glacialis Arctic Ocean predictions for the year 2100 (pHNBS 7.6) left EHS unchanged, although high var-
iation within treatments may have masked effects [36]. When subjecting C. finmarchicus
females and eggs to pHNBS 6.95, EHS was strongly reduced [53], but in the congener C. helgo-
landicus EHS did not change when subjecting the copepods to pHNBS 7.75 [54]. Considering
other calanoid genera, the picture looks somewhat different. EHS was significantly lowered in
Acartia clausi at pHT 7.83 compared to pHT 8.03 [55]. Similarly, in Acartia tonsa EHS was sig-
nificantly lowered already at pHNBS 7.81 [56]. However, the copepods for the A. tonsa study
were obtained from commercial cultures in which the carbonate chemistry may be different
than in the wild. It may well be that copepods from such cultures could be adapted to an envi-
ronment with only minor chemical variations and therefore would respond stronger to pH
changes. In Acartia steueri, EHS was significantly depressed at pH 7.3 (no pH scale indicated)
when comparing effects through three generations [57]. However, when trying to extract spe-
cific information on the effects of long term multigenerational exposure the significant differ-
ence disappeared, although this may have been due to low statistical power. PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
February 7, 2018 Supporting information S1 Table. Raw values of unhatched eggs remaining versus time. The first sheet, “info”, con-
tains information on the ordering of the buckets and replicate flasks in the data sheets. Data
sheets are arranged according to target buckets in the transplant (H meaning high pH and L
meaning low pH). In each sheet the first column shows origin bucket label, the second column
shows target bucket label, and the following columns show time (d) and number of unhatched
eggs remaining at each sampling event. (
) Ocean acidification effects in Calanus glacialis et al. data [60] reveals that samples were not taken simultaneously around Km for the different
treatments. This non-simultaneous sampling may have biased results and there could be a risk
that differences in the sigmoid accumulation of hatched eggs among treatments emerged as an
artefact. Effects of OA levels predicted for year 2100 on egg hatching seem to be lower in larger than
smaller calanoid copepods. Such difference could arise from differences in the sensitivity of
energy allocation to OA between the two groups. For instance, energetic expenses of growth
seems to increase at decreasing pH in female P. acuspes [48], and a recent study indicated that
this may also be true for C. glacialis stage IV copepodites [46]. On the other hand, a lack of sim-
ilar effects in stage V copepodites in that same study showed that effects may be stage depen-
dant and adult females may respond differently [46]. Alternatively, the differences may stem
from differences in ontogeny. Copepods of the large Calanus genus undergo ontogenetic verti-
cal migration and winter hibernation in the deep. During such overwintering, the copepods
experience haemolymph pH possibly as low as 5.5 as a result of metabolic depression during
hibernation [64]. C. glacialis is an opportunistic capital breeder and production of the first
eggs of the season often takes place in the deep [41]. It is therefore quite conceivable that mech-
anisms to counter low pH could have evolved to protect early oogenesis. Later in the season,
eggs are predominately produced on ingested energy (income breeding) [40, 41]. Broader sup-
port for the idea that taxa living under different pH regimes have evolved different physiolo-
gies has been found also in pteropods and polychaetes [65–68]. Naupliar growth and development is similarly unaffected and it seems that C. glacialis will
develop from oviposition to the first copepodite stage unaffected under the future OA scenario
tested here [33]. Consequently, OA will not alter the exposure to early life predation mortality
or introduce any phenological miss-match with phytoplankton bloom timing. Similarly, larval
growth and development of the congener C. finmarchicus is equally unaffected which adds
strength to the argument that naupliar recruitment and development in the Calanus genus is
generally unaffected by OA [69]. Acknowledgments We thank the staff at the Kings Bay Marine Lab, Ny-Ålesund for their invaluable technical
assistance and the staff at the Sverdrup Station, Ny-Ålesund for logistic support. Discussion While increasing temperatures may at least partially remove the ice
algal bloom, they also shifts the pelagic bloom to occur earlier in the season. Copepods experi-
ence the same shift, but it not as pronounced resulting in a temporal trophic miss-match,
which impairs optimal exploitation of the bloom [25]. Obviously, any delay in egg hatching by
OA would only accentuate this miss-match. Hatching rate has been shown to be significantly
impaired already at pHT 7.75 in Pseudocalanus acuspes [49]. Also, studies on Acartia sp. have
shown significant delay of egg hatching at low pH [60]. However, a closer look at the Vehmaa 7 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
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Jacque, Piero Calosi. Writing – original draft: Peter Thor, Fanny Vermandele, Marie-Helene Carignan, Sarah
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l Formal analysis: Peter Thor, Fanny Vermandele, Marie-Helene Carignan, Sarah Jacque, Piero
Calosi. 8 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0192496
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Gross Motor Levels in Early Childhood Post-Online Learning in the Highlands Region: Cross Sectional Study
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Abstract Gross motor development in children aged 4-5 years is an important aspect to support children's growth and
development, in the learning phase after the pandemic and curriculum changes require an evaluation process of
gross motor skills using a gross motor ability survey method in children after 4-5 years in Stone City. The subjects
in this study totaled 164 students who were taken from 9 kindergartens/PAUD in Batu City. The sampling protocol
in this study used the cluster random sampling method. The instruments used in this study used the components
in the active motor card, which included: 1) gallop, 2) hop, 3) slide, 4) s-tug, 5) catch, 6) kick, 7) overhand throw,
8 ) underhand roll. The average result of gross motor skills in boys is 51.98 in the "good" category and in girls it
has an average of 52.92 in the "good" category. The gross motor ability test results for children aged 4-5 years in
Batu City adjusted to the age and characteristics of children. The gross motor skills of children aged 4-5 years are
better in locomotor motion instruments so that in this case the motion of using tools or objects can be implemented
more by educators to stimulate children's gross motor motor skills. Keywords: Gross Motor, Preschooler, Highland Received: 25 May 2023
Revised: 5 July 2023
Accepted: 11 Agust 2023
Published: 30 September 2023 Gross Motor Levels in Early Childhood After Online Learning in
the Highlands Region: Cross Sectional Study
Eldi
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i di1 Eldiene Zaura I’tamada*1, Mashuri Eko Winarno2, Imam Hariadi1 Bravo’s: Jurnal Program Studi Pendidikan Jasmani dan Kesehatan
Volume 11 Number 3, September 2023, pp: 248-258
E-ISSN: 2597-677X; P-ISSN: 2337-7674
DOI: http://dx.doi.org/10.32682/bravos.v11i3/3011 INTRODUCTION The importance of physical activity as one of the main forms of health for all levels of
society has been strengthened by the guidelines set by WHO. Specifically, the benefits of
physical activity for children and adolescents as having a significant impact on metabolic
systems (cardiovascular fitness, blood pressure, and glucose), cognitive (academic
performance, and executive function) and mental health (reduced depressive symptoms) are
summarized, and it is recommended that Children need at least 60 minutes per day of moderate
to high intensity physical activity throughout the week (World Health Organization, 2020b). Physical inactivity is a global public health problem primarily associated with various chronic
disease outcomes (Hall et al., 2020). Thus, the motivations and barriers underlying whether a
person engages in physical activity or not are of critical public health interest (Hoare et al.,
2016, 2017; Vera et al., 2018). To improve this requires simultaneous cooperation from each
country that has been regulated by supporting organizations. The Active Healthy Kids Global
Alliance is an organization that focuses on the physical activity of children and adolescents
throughout the world. To encourage this level of physical activity there are several indicators
of physical activity in children which have been explained and monitored in a document called
a "Report card" which was created as a tool for physical activity advocacy (Active Healthy
Kids Global Alliance, 2018). Meanwhile, 5-25% of young children experience developmental 248 Gross Motor Levels in Preschool Children in Low-Middle Income Countries in the Highlands Region: Cross Sectional Study disorders, with 8 to 9% of young children experiencing problems with the child's psychomotor
development and gross motor skills (World Health Organization, 2020a). The current post-
pandemic condition is also a major factor in decreasing motor skills and lack of physical
activity for children (Ayubi & Komaini, 2021; Pardilla et al., 2020), identification of children's
motor skills is very important to implement. COVID 19 has a significant effect on endurance,
motor ability and immunity (Amatriain-Fernández et al., 2020; da Silveira et al., 2021; Dwyer
et al., 2020). One of the factors that influences the level of endurance, and the immune system
is the composition of the movement behaviour. Children's activities are limited during the
pandemic. Children's visits and interactions with playgrounds and schools are limited. INTRODUCTION Movement activities are limited, stress levels increase and children's boredom increases,
making them vulnerable to a decline in the immune system (Jiao et al., 2020; Moore et al.,
2020). The 2021 PAUD and PNF Ban policies and mechanisms regarding children's physical
and motor development explain that at least every school has a teacher or facilitator who
understands children's movement needs. This requires policies to support physical activity in schools and in the larger community
through education, the built environment, and the use of tested and programmed curricula. Healthy physical activity patterns built from childhood can support a healthy lifestyle and must
be maintained throughout life (Peyer, 2021). In this context, aspects of social and economic
change in countries with a high human development index urge action to increase physical
activity among children and adolescents. Supervision and monitoring efforts are needed to
identify gaps in the coaching process so that it requires development that focuses on the results
of children's physical evaluations (Aubert et al., 2018). These activities need to be supported
by government policies as well as the school environment, community, and family environment
regarding supporting programs to develop systems to increase physical activity in children, use
of active transportation, active play patterns in peer, family and school environments and
reduce sedentary time spent every day (Aubert et al., 2018). This can be obtained from school
data and the achievement of student movement indicators developed in an area. However,
fulfilling physical activity in children requires movement literacy that supports the habituation
process. The issue of physical activity and children's motor skills is still something that receives
little attention (Liu & Chen, 2021). Movement literacy in children will form awareness
regarding the importance of physical activity. which will influence the thinking patterns of the
younger generation, become a lifestyle and be able to develop personal potential and healthy
living habits. The recommended pedagogical strategies are related to physical literacy in 249 Eldiene Zaura I’tamada, Mashuri Eko Winarno, Imam Hariadi children according to their age phase to develop children's potential and interests which not
only increase competence but also focus on children's movement performance (Lundvall,
2015). So, in this case, to determine the motor skills of young children, a test is needed as an
effort to determine gross motor surveillance in children aged 4-5 years in Batu City. METHOD The research method used was a survey using a cross-sectional approach by conducting
gross motor skills tests with locomotor test instruments and control objects from the active
motor card which contains 8 instruments, which include: 1) gallop, 2) hop, 3) slide, 4) s-tug,
5) catch, 6) kick, 7) overhand throw, 8) underhand roll adopted from Test Gross Motor
Development-2 (TGMD-2) (Ulrich & Sanford, 2000). The sampling protocol used the cluster
random sampling method which was carried out at 9 schools in Batu City with a total of 164
students as subjects. Data were analysed using a quantitative approach by identifying and
describing data in the form of statement sentences regarding data on gross motor skills of
children aged 4-5 years. This research has passed the ethical feasibility test determined by the
Malang POLKESMA ethics commission with certificate number: 620/KEPK-POLKESMA/
2022. INTRODUCTION The results
of evaluating children's motor skills can be developed to provide recommendations for
children's physical needs, so that with the evaluation process early childhood development will
be well recorded. This research focuses on the gross motor skills of children aged 4-5 years
because motor development is an important aspect in developing skill processes and movement
patterns that children carry out using the whole body (Sukamti, 2018). RESULTS AND DISCUSSION The test results in this research can be used as a learning evaluation process to determine
the gross motor development of early childhood children on a regional scale. This research was
conducted on children aged 4-5 years (kindergarten class A) which was carried out only to
determine the children's gross motor skills. The results of the gross motor skills test for children
aged 4-5 years using the instruments on the Active Motor Card used in Batu City can be seen
in detail in table 1. The average gross motor skills of children aged 5 years are greater than
children aged 4 years. At the age of 4 years, boys tend to have better grades than girls and at
the age of 5 years, girls have greater grades and tend to be the same as boys. The results of the
child's abilities on each instrument are described in figures 1 to 4 which explain according to
the child's age and gender. The figure explains that boys aged 4-5 years and girls aged 4 years
tend to have better scores on the locomotor test, girls aged 5 years have relatively similar motor 250 Gross Motor Levels in Preschool Children in Low-Middle Income Countries in the Highlands Region: Cross Sectional Study ability results between the locomotor test and the control object. The results of gross motor
skills are compared in table 2 and table 3. The results of gross motor skills in preschool children were obtained through tests of 1)
gallop, 2) hop, 3) slide, 4) s-tug, 5) catch, 6) kick, 7) overhand throw, 8) underhand roll which
were grouped according to age and gender of the child. Each instrument has a movement
category that is intended to assess the child's movement process. From the tests carried out,
each instrument is added up and a conclusion is given from all the tests carried out. So, after
testing 164 children, the results of the children's gross motor skills included: Table 1. Descriptive data on gross motor skills of children aged 4-5 years
Kelompok
N
Means
Std. Deviation
Minimum Maximum
4-year-old male
9
52,00
4,90
42
60
5-year-old male
80
53,99
5,55
31
63
4-year-old female
10
51,00
4,71
43
57
5-year-old female
65
54,14
6,78
22
64
Total
164
53,76
6,05
22
64 Table 1. Descriptive data on gross motor skills of children aged 4-5 years
Kelompok
N
Means
Std. RESULTS AND DISCUSSION Deviation
Minimum Maximum Table 1. Descriptive data on gross motor skills of children aged 4-5 years The results of the motor skills of children aged 4-5 years in 9 kindergartens in Batu City
which have been adjusted to the active motor card norms which are adjusted to the child's age
and gender, included: The results of the motor skills of children aged 4-5 years in 9 kindergartens in Batu City
which have been adjusted to the active motor card norms which are adjusted to the child's age
and gender, included: 7
3
5
7
3
6
3
2
0
3
2
0
2
0
3
4
0
2
1
0
4
2
2
2
0
0
0
0
0
0
0
0
2
1
1
2
0
1
1
1
0
1
2
3
4
5
6
7
8
Gallop
Hop
Slide
S-Tug
Catch
Kick
Overhand
Throw
Underhand Roll
SB
B
C
K
KS
Figure 1. Results of Motor Ability for 4-Year-Old Male SB
B
C
K
KS SB
B
C
K
KS
Figure 1. Results of Motor Ability for 4-Year-Old Male The total sample of 4-year-old children with male gender was 9 children, the results of
gross motor skills in the very good category were gallop, slide, s-tug and kick movements. In
this movement, 7 children had very good motor skills and 2 other children were in the very
poor category. Of the 8 existing instruments, the results of children's gross motor skills tend to
be in the very good, good, and fair categories, so this instrument tends to be used for boys aged
4 years. 251 Eldiene Zaura I’tamada, Mashuri Eko Winarno, Imam Hariadi 0
34
0
0
49
0
27
34
68
25
68
73
15
62
43
9
5
12
5
0
14
4
4
27
0
0
0
0
0
0
0
0
7
9
7
7
2
14
6
10
0
10
20
30
40
50
60
70
80
Gallop
Hop
Slide
S-Tug
Catch
Kick
Overhand
Throw
Underhand
Roll
SB
B
C
K
KS
Figure 2. Results of Motor Ability for 5-Year-Old Male SB
B
C
K
KS Figure 2. Results of Motor Ability for 5-Year-Old Male Figure 2. RESULTS AND DISCUSSION Results of Motor Ability for 5-Year-Old Male The results of the gross motor skills of 5-year-old boys have an average of good
categories on the gallop and slide instruments with a percentage of 85% of children getting the
good category, s-tug with a good percentage of 91.25%, kick with a good category percentage
of 77 .5%, and 53.75% in the good category on the overhand throw instrument. The results in
the very good category were more numerous for the hop instrument with a percentage of 42.5%
and 61.25% for the catch instrument. 4
3
0
5
4
4
4
1
3
4
8
2
2
4
1
7
3
2
1
2
4
0
4
1
0
0
0
0
0
0
0
0
0
1
1
1
0
2
1
1
0
1
2
3
4
5
6
7
8
9
Gallop
Hop
Slide
S-Tug
Catch
Kick
Overhand
Throw
Underhand
Roll
SB
B
C
K
KS
Figure 3. Results of motor skills of 4-year-old female SB
B
C
K
KS Figure 3. Results of motor skills of 4-year-old female Figure 3. Results of motor skills of 4-year-old female For children aged 4 years and female, the results tend to be in an even category between
very good, good, and fair, while children who get the category are very poor with 1 child on
several instruments. The average results in the good category for slide and underhand roll
instruments tend to be higher than for other instruments. The number of good and very good
categories on the gallop, hop and s-tug instruments has a total of 7 children. 0
28
0
0
33
35
29
21
53
28
57
55
7
11
5
23
8
5
4
6
23
12
29
20
0
0
0
0
0
0
0
0
4
4
4
4
2
7
2
1
0
10
20
30
40
50
60
Gallop
Hop
Slide
S-Tug
Catch
Kick
Overhand
Throw
Underhand Roll
SB
B
C
K
KS
Figure 4. Results of Motor Ability of 5 Year Old Female Figure 4. RESULTS AND DISCUSSION Results of Motor Ability of 5 Year Old Female 252 Gross Motor Levels in Preschool Children in Low-Middle Income Countries in the Highlands Region: Cross Sectional Study The results for girls aged 5 years had an average good category on locomotor instruments,
with 53 children on the gallop instrument, 57 children on the slide instrument, 55 children in
the s-tug category, and 28 children in the excellent category. with a good category on hop
instruments. Basic movements such as gallops, slides, and coordination without objects such
as s-tugs will be easier for children to do, because preschool-aged children master more basic
locomotor movements than games (Ruiz-Esteban et al., 2020). In preschool children, activities
with basic locomotor movements such as coordination and balance carried out twice a week
can develop children's gross motor skills, so that the main thing that is done in the introduction
of movement in educational institutions is to develop children's locomotor abilities first (Costa
et al., 2015; Iivonen & Sääkslahti, 2014). Table 2. Gross Motor Test Results for Children Aged 4-5 Years
Norm Reference
Very Well
Well
Enough
Less
Very Less
4 Years Old Male
2
4
2
0
1
Relative (%)
22.2
44.4
22.2
0
11.1
5 Years Old Mael
34
9
27
0
10
Relative (%)
42.5
11.25
33.75
0
12.5
4 Years Old
Female
1
7
1
0
1
Relative (%)
10
70
10
0
10
5 Years Old
Female
21
23
20
0
1
Relative (%)
32.3
35.8
30.76
0
1.53 Table 2. Gross Motor Test Results for Children Aged 4-5 Years The results of the gross motor skills test for boys aged 4-5 years in Batu City with a total
of 86 students had an average test result of 51.98 in the good category, 30 students got the very
good category, 32 students got the good category, 18 students in the sufficient category and 6
students in the very poor category. The test results for girls aged 4-5 years were 26 students in
the very good category, 32 students in the good category, 12 students in the fair category and
5 students in the very poor category. The total number of female students was 75 students with
an average result of gross motor skills of 52.92 which was in the good category. RESULTS AND DISCUSSION These results
do not have a significant difference between boys and girls, boys and girls aged 0-6 years do
not have significant differences in their motor abilities. In terms of daily outdoor activities
carried out by girls, they tend to take longer than boys but still tend to be the same and not
significantly different, the activities carried out tend to involve more movement of control
objects (Kwon et al., 2022). Locomotor movements which tend to be basic have their own
difficulties related to the movement process carried out by children. In the process of
developing their movements, the implementation of learning by doing movements which are
carried out in a fun way is required (Calero-Morales et al., 2023). Basically, boys and girls at 253 Eldiene Zaura I’tamada, Mashuri Eko Winarno, Imam Hariadi an early age have different neurobiological mechanisms based on gender, different mental
development processes in boys and girls have the same influence on their cognitive processes
(Fernández‐Sánchez et al., 2022). In this case, early childhood gross motor development is very
important as a supporting process for children's development. The evaluation process in physical education emphasizes the child's gross motor
movement process with the aim of knowing the development of the body, and the results of the
movement recognition process carried out so that it has benefits on growth and development. With the participation of students in carrying out physical activities, neuromuscular skills will
develop and influence the student's motor movement mechanisms. This is very necessary to
improve physical activity skills which have an impact on social aspects. There are five basic
biomotor skills that are important for children's growth and development, including: strength,
endurance, coordination, speed, and flexibility. These five basic movements can be developed
into other movement elements, such as explosive power, agility, endurance, beauty of
movement and so on (Balyi et al., 2013; Bompa & Buzzichelli, 2019). The instruments
developed to determine the gross motor skills of young children use the TGMD-2 (Test Gross
Motor Development-2). The TGMD-2 has 12 instruments which are used by dividing the
components into two parts which include locomotor and object control (Ulrich & Sanford,
2000). RESULTS AND DISCUSSION The instruments in TGMD-2 have some movement difficulties when used for children
aged 4-5 years, some easy movements are related to kicking, running, jumping and sideways
(Kezić et al., 2020) but some other instruments tend to be more difficult , aspects related to the
instrument must be adjusted to the child's characteristics, because the child's gender, habits,
lifestyle and environment influence their characteristics (Webster et al., 2019). The test results
for children in the highlands tend to have lower motor skills than children in coastal areas with
results of 54.1% in the very superior category, 37.5% in the superior category, 4.2% in the
above average and average categories. , in the highlands it has a value of 9.5% in the average
category, 28.6 in the bellows average and poor category and in the very poor category with a
percentage of 33.2% (Samodra et al., 2023) because basically the ability of children in each
region will differ depending on the habits and culture of the area, so in this case the motor skills
test is carried out using instruments that have been adapted to the child's condition. Batu City
has a relatively high regional income at number 8 in 2022 in Indonesia, in this case the motor
skills of children in areas with low income are less likely to experience iron deficiency,
anaemia, risk of gross motor and communicative delays (Celhay et al., 2020). In this case,
development progress and the economic level which tends to be high in Batu City have a quite 254 Gross Motor Levels in Preschool Children in Low-Middle Income Countries in the Highlands Region: Cross Sectional Study significant impact on the gross motor skills of children aged 4-5 years which can be seen from
the test results which tend to be in the good category and children aged 4-5 years are easier. make locomotor movements rather than penis objects even though basically children prefer to
play using tools. CONCLUSION The results of the gross motor skills test for children aged 4-5 years in Batu City using
the test instrument on the active motor card have an average of good category. The use of
evaluation instruments used in early childhood can be done by adjusting the child's condition,
region, and child's age. So, this research uses instruments on active motor cards which have
been adapted to early childhood in Batu City. Measuring gross motor skills in early childhood
can use the test gross motor development-2 (TGMD-2) instrument but using this instrument in
several categories tends to be too difficult to carry out in children aged 4-5 years (TK A) and
the tools used in These instruments are difficult to find in early childhood school institutions. So, by using a battery test that has been adapted to the child's condition according to the region,
it can simplify the process of evaluating a child's gross motor skills. On the gross motor skills
test results, boys have a lower average than girls, but these results are still considered good and
do not have a significant difference. The use of test instruments to determine children's motor
skills is adjusted to the age and socio-demographic characteristics of children in Batu City. The
results of tests on gross motor skills in children aged 4-5 years are better on locomotor
movement instruments so that in this case movements using tools or objects can be
implemented more by educators to stimulate children's gross motor skills. This research was
only carried out on children aged 4-5 years (kindergarten class A) at an early childhood
institution which will switch to class B. So, in this case future research can be carried out on
children aged 5-6 years (kindergarten class B) with aspects of children's gross motor and fine
motor skills because currently post-covid learning conditions in early childhood have limited
motor ability evaluation instruments which have not been redesigned by early childhood
institutions. With this, educators need instruments that can be used to determine children's
development and growth so that this research can make it easier for educators to find out the
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https://openalex.org/W4319710815
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https://juah.uoanbar.edu.iq/article_174755_288296918bd65080f0376ce41a5754e5.pdf
|
Arabic
| null |
Occupation and Profession in the Book ((GhayaT Al-Nahiya fi Tabaqat Al-Reader)) by Ibn Al-Jazari Died 833 A.H
|
Deleted Journal
| 2,022
|
cc-by
| 11,572
|
المجمد19
-
العدد2
–
حزيران
2022
Volume 19- Issue 2- June 2022
الوظيفة والمهنة في كتاب ((غاية النهاية في طبقات القراء)) البن الجزري ت333هـ
.أ.د
فاطمة زبار عنيزان
جامعة بغداد-
مركز
احياء التراث العممي العربي
Fatema.za@gmail.com
الممخص:
DOI
10 37653/juah 2022 174755 المجمد19
-
العدد2
–
حزيران
2022
Volume 19- Issue 2- June 2022
الوظيفة والمهنة في كتاب ((غاية النهاية في طبقات القراء)) البن الجزري ت333هـ
.أ.د
فاطمة زبار عنيزان
جامعة بغداد-
مركز
احياء التراث العممي العربي
Fatema.za@gmail.com
الممخص:
DOI
10 37653/juah 2022 174755 DOI
10.37653/juah.2022.174755 الممخص: اتبع ابف الجزري منيجا قائما عمى أساس عرض وتحميؿ ما لو مف
عالقة بوظيفة مترجمو ومينتو في كتابو ((غاية النياية في طبقات
القراء))،وىذا المنيج عكس لنا اىتمامو العاـ بالتراجـ فقد حرص عمى
إيرادىا بأدؽ تفاصيميا وأوسع المعمومات عنيا،إذ عرضيا ضمف االسـ
متبع في ذلؾ اتجاىات عدة وربطيا بشكؿ متداخؿ مع المعمومات األخرى
الخاصة بالترجمة سواء كانت موسعو أو مختصرة ،وجعميا مرتبطة بشكؿ
مباشر مع األحداث التي كانت متداخمة في الترجمة وعرضيا بأسموب
إداري أو عممي أو حرفي وغيرىا مف األحواؿ التي كانت عمييا البالد
.العربية واإلسالمية (
289
) Occupation and Profession in the Book ((GhayaT Al-Nahiya fi Tabaqat
Al-Reader)) by Ibn Al-Jazari Died 833 A.H Submitted: 05/01/2022
Accepted: 28/03/2022
Published: 01/06/2022 Abstract: He followed an approach based on the presentation and
analysis of his money related to his translator’s job and
profession. Or abbreviated, and make it directly related
to the events that were intertwined in the translation and
presented in an administrative, scientific or literal
manner and other conditions in which the Arab and
Islamic countries were. Keywords: Job and profession
Ghayat Al-Nahiya book
Ibn Al-Jazari. Job and profession
Ghayat Al-Nahiya book
Ibn Al-Jazari. ©Authors, 2022, College of
Education
for
Humanities
University of Anbar. This is
an open-access article under
the
CC
BY
4.0
license
(http://creativecommons.org/li
censes/by/4.0/). (
289
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
المقدمة هلااهل جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
ة جملة جامعة االنبار للعلوم االنسانية الحمد هلل رب العالميف والصالة والسالـ عمى سيدنا محمد "صمى اهلل ع "ميو وسمـ
وعمى الو وصحبو أجمعيف. أما بعد يعد ىذا النوع مف الدراسات ألميمة في مجاؿ البحث العممي القائـ عمى أساس
تتبع
الوظيفة والمينة وب تحميؿ ظاىرة معينة مف تمؾ الظواىر التي تقع في كتب التراجـ السيما
إنيا تشكؿ إحدى المواد األساسية لموارد ىذا الكتاب ، وبالتالي تقع عمى الباحث ميمة
التحميؿ في مثؿ ىذا الموضوع ألنو قائـ عمى أساس التنوع والدقة والغموض ، ويحتاج إلى
جيد كبير مف اجؿ الوصوؿ إلى ىدفو ، لذا فاف مدى أو تنوع الطرؽ التي اعتمدىا ا بف
الجزري في اقتباس نصوصو مف مصادرىا شكؿ احد العناصر األساسية التي يبيف لنا مدى
الشموؿ المنيجي لموارد كتابو "غاية النياية في طبقات القراء" ، تستحؽ مف الباحث الدراسة
والتحميؿ لموصوؿ إلى مدى التطور الذي كاف عميو منيج ابف الجزري خالؿ مف التعامؿ مع
النصوص وتحم.يميا في الوظيفة والمينة موضوع دراستنا ليذا البحث المبحث األول: ابن الجزري: السيرة والمكانة العممية المبحث األول: ابن الجزري: السيرة والمكانة العممية أ
أوال: سيرة ابن الجزري أوال: سيرة ابن الجزري أوال: سيرة ابن الجزري 1
-
:اسمه ولقبه 1
-
:اسمه ولقبه احمد بف محمد بف محمد بف محمد بف عمي بف يوسؼ بف الجزري أبو بكر(
1
)
، أبو
الخير شمس الديف العمري الدمشقي ثـ الشيرازي الشافعي(
2
)
، أو كما أورده السخاوي احمد بف
محمد بف محمد بف عمي بف يوسؼ الشياب أبو بكر(
3
). احمد بف محمد بف محمد بف محمد بف عمي بف يوسؼ بف الجزري أبو بكر(
1
)
، أبو
الخير شمس الديف العمري الدمشقي ثـ الشيرازي الشافعي(
2
)
، أو كما أورده السخاوي احمد بف
محمد بف محمد بف عمي بف يوسؼ الشياب أبو بكر(
3
). ولقب بالجزري نسبة إلى الجزيرة مف ديار بكر(
4
)
، وىناؾ ألقاب أخرى تميز بيا
فضالً عف ىذا منيا: أبو الخير(
5
)، شمس الديف(
6
)، شياب الديف(
7
). Keywords: ثان
ياً: مكانته العممية ثان
ياً: مكانته العممية 1
-
:ثقافته Keywords: وىناؾ بعض األلقاب التي أسبغت عمى اسمو غمب
عمييا الطابع والدرجة العممية التي
كاف عمييا ابف الجزري منيا: الحافظ(
8
) ، المقرئ(
9
) ، شيخ اإلقراء(
10
)، العالّمة(
11
)
، المتكمـ
والحجة(
12
) ، الشيخ(
13
). ولقب بالجزري نسبة إلى الجزيرة مف ديار بكر(
4
)
، وىناؾ ألقاب أخرى تميز بيا
فضالً عف ىذا منيا: أبو الخير(
5
)، شمس الديف(
6
)، شياب الديف(
7
). وىناؾ بعض األلقاب التي أسبغت عمى اسمو غمب
عمييا الطابع والدرجة العممية التي
كاف عمييا ابف الجزري منيا: الحافظ(
8
) ، المقرئ(
9
) ، شيخ اإلقراء(
10
)، العالّمة(
11
)
، المتكمـ
والحجة(
12
) ، الشيخ(
13
). )
984
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
2
-
:والدتــه
ولد ابف الجزري في ليمة الجمعة السابع عشر مف رمضاف سنة ثمانيف وسبعمائة
(( بدمشؽ كما ورد في كتابو غاية النياية في طبقات القراء)) وىي الرواية األصح
بتقديرنا(
14
)
، إال أف ىناؾ تضارب في بعض الروايات األخرى حوؿ والدتو ، منيا انو ولد سنة
إحدى وخمسيف وسبعمائة(
15
)
، وأخرى انو ولد بدمشؽ ليمة السبت الخامس والعشريف مف شير
رمضاف سنة إحدى وخمسيف وسبعمائة(
16
). جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 و ا
ولد ابف الجزري في ليمة الجمعة السابع عشر مف رمضاف سنة ثمانيف وسبعمائة
(( بدمشؽ كما ورد في كتابو غاية النياية في طبقات القراء)) وىي الرواية األصح
بتقديرنا(
14
)
، إال أف ىناؾ تضارب في بعض الروايات األخرى حوؿ والدتو ، منيا انو ولد سنة
إحدى وخمسيف وسبعمائة(
15
)
، وأخرى انو ولد بدمشؽ ليمة السبت الخامس والعشريف مف شير
رمضاف سنة إحدى وخمسيف وسبعمائة(
16
). في
الوقت الذي كاف ىناؾ تضارب في بعض الروايات حوؿ والدة ابف الجزري ، فمـ
يكف ىناؾ تضارب حوؿ وفاتو في بعض الروايات ، فقد توفي ابف الجزري ضحى يوـ
الجمعة لخمس خموف مف أوؿ الربيعيف سنة ثالث وثالثيف وثمانمائة بمدينة شيراز ودفف بدار
القرآف التي أنشاىا وكانت جنازتو
مشيورة(
17
)
، وقد تبادر األشراؼ والخواص والعواـ إلى
حمميا وتقبيميا ومسيا تبريكا(
18
)
، ومف لـ يمكنو الوصوؿ إلى ذلؾ كاف يتبرؾ بمف تبرؾ بيا
وأكد ذلؾ الشيخ محمد عمي الضباع (
19
) ، في مقدمة شرح كتاب طيبة النشر (
20
)
، وقد
أندرس بموتو كثير مف المياـ العممية(
21
). 1
-
:ثقافته عني ابف الجزري بطمب العمـ ، السيما انو نشأ وتربى وتمقى العمـ في صغره عمى يد
والده المعمـ والمربي األوؿ البف الجزري ، ورغبتو الصادقة في ذلؾ فضالً عف رحالتو الكثيرة
ومالزمتو لعمماء عصره كاف لو اثر في تكويف شخصيتو العممية، فأصبح
ذا شأف كبير إذ برع
:ًفي عموـ عدة ، فيشير الحسيني انو كاف إماماً في القراآت وغيرىا مف العموـ األخرى قائال
"..."...وكاف إماماً في القراآت ال نظير لو في عصر الدنيا حافظاً لمحديث وغيره(
22
)
، وأشاد
السيوطي بعممو والعموـ التي برع بيا إال انو آخذ عميو عدـ معرفتو
بالفقو كما يقوؿ: "...وبرع
في القراآت... وكاف إماـ في القراآت ال نظير لو في عصره في الدنيا حافظاً لمحديث وغيره
"أتقف فيو ... ولـ تكف لو في الفقو معرفة(
23
)
، وأشاد بو ابف العماد وبعممو عمى مستوى األمة
اإلسالمية وبفصاحتو وبالغتو قائالً: "...مقرئ الممالؾ اإلسال ًمية وكاف شكالً حسناً مثريا
"...ًفصيحاً بميغا(
24
). )
985
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 ( العدد2
( ) اجمللد91
)
حزيران
2222 تمقى ابف الجزري عمومو عمى عدد مف الشيوخ الذيف كاف ليـ دور كبير في صقؿ
شخصيتو العممية والوصوؿ إلى ما ىو عميو مف عمـ وم
عرفة ورفعة ومكانة عالية في عصره ،
وكاف والده أوؿ شيوخو الذيف نيؿ منيـ العمـ كو نو المعمـ والمربي األوؿ الذي أحاط بو ورعاه
مما لو أثره عمى سيرتو العممية ، كما يقوؿ عنو "...و حفظ ... قصدني في العشر ولما رحمت
بأخيو لقراءة القراآت عمى ابف العسقالني آخر أصحاب التقي الصايغ قرأ معو عميو قطعة مف
أوؿ القرآف... وأكمؿ عمى أيضاً القرآف بالقراآت "العشر وقرأ عمى كتابي النشر و "التقريب
"...والطيبة(
25
)
، وأشاد بو والده ألنو شرح منظومتو في غيابو قائالً: "... 1
-
:ثقافته قرأت معو
"...عميو قطعة مف أوؿ القرآف(
31
)
:ً، ويزيد السخاوي انو سمع عمى ابف العسقالني قائال
"..."...ومما سمعو عمى ابف العسقالني جميع القراآت(
32
)
، وقرأ أيضاً عمى إبراىيـ بف احمد
الحريري (ت800
")ىػ(
33
)
الذي أجازه كما يقوؿ: "...ثـ أقرأ فقرأ عميو...ابف أبو بكر
"حدثي بالقراآت أيضاً عف جماعة باإلجازة
احمد(
34
) )
986
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
وىذا يدؿ عمى سعة عممو واختالؼ معارفو ومنابع عممو األصمية وتعدد عممائو الذيف
،أخذ عمييـ لو دور في بناء شخصيتو العممية والفضؿ في ذلؾ يعود لوالده الذي اىتـ بو
.ووجيو الوجية الصحيحة واألولى في تمقي العموـ
3
-
:تالمذته ( العدد2
( ) اجمللد91
)
حزيران
2222 جملة جامعة االنبار للعلوم االنسانية جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
وىذا يدؿ عمى سعة عممو واختالؼ معارفو ومنابع عممو األصمية وتعدد عممائو الذيف
،أخذ عمييـ لو دور في بناء شخصيتو العممية والفضؿ في ذلؾ يعود لوالده الذي اىتـ بو
.ووجيو الوجية الصحيحة واألولى في تمقي العموـ
3
-
:تالمذته وىذا يدؿ عمى سعة عممو واختالؼ معارفو ومنابع عممو األصمية وتعدد عممائو الذيف
،أخذ عمييـ لو دور في بناء شخصيتو العممية والفضؿ في ذلؾ يعود لوالده الذي اىتـ بو
.ووجيو الوجية الصحيحة واألولى في تمقي العموـ
3
-
:تالمذته مثمما نيؿ ابف الجزري العموـ المختمفة مف المشايخ واألساتذة ، كاف لو عدد مف
التالميذ نيمو منو العموـ المختمفة سواء بالسماع أو القراءة أو اإلجازة ، منيـ عمى سبيؿ ا لمثاؿ
ال الحصر: إبراىيـ بف احمد الطباطبي "سمع عمى ابف الجزري جميع األربعيف
"...النووية(
35
)
، واحمد بف عمي بف عمر " قرأ عمى ابف الجزري طيبتو مف حفظو وأجاز
"لو(
36
)
"...، واحمد بف محمد البنجوري "سمع عمى ابف الجزري(
37
)
، وشاىيف المنصوري "سمع
..عمى ابف الجزري الشفاء". (
38
)
، وعمى بف محمد األنصاري "...قرأ ابف الجزري مشيختو
"الفخر(
39
)
، وعمر بف عبد الحميد المدني "...سمع عمى ابف الجزري الشفا في سنة ثالث
"...وعشريف وثمانمائة وضبط األسماء(
40
)
، ومحمد بف إبراىيـ الخجندي "...قرأ األربعيف
بتماميا في مجمس واحد عمى ابف الجزري في ربيع اآلخر سنة ثالث وعشريف بالحرـ
"...النبوي(
41
)
"...، والحسيف حمزة بف عمي "... 1
-
:ثقافته ولما كاف بمصر
بمغني وأنا مجاور لمكة شرح طيبة النشر فأحسف فيو ما شاء اهلل مع انو لـ يكف عنده نسخة
بالحواشي التي كتبتيا عميو ومف قبؿ ذلؾ شرح مقدمة ا لتجويد ومقدمة الحديث مف نظمي في
"...غاية الحسف(
26
)
، ومف شيوخو اآلخريف نذكر منيـ عمى سبيؿ المثاؿ ال الحصر وكاف
:ليـ األثر األكبر في تكويف شخصيتو العممية: الصالح بف االعزازي الذي اخذ منو كما يقوؿ
"...فأدرؾ الصالح بف محمد بف احمد بف أبي عمر آخر أصحاب البخاري"...وأجازه(
27
)
،
:وأشار إلى عدد مف الشيوخ والمسنديف الذيف أجازوه لعمو منزلتو العممية ، كما يقوؿ عنو والده
"...وكذلؾ أجازه المشايخ والمسندوف إذ ذاؾ كالقاضي ابف شيبة وابف عوض والتاج محبوب
"...وابف السالر والحافظ ابف المحب وحضر عندىـ وسمع مف آخريف(
28
)
، وأخذ مف الصالح
محمد بف عمر الصامت (ت789
ىػ) ، كما يقوؿ عنو: "...وحدثني بكثير مف مسموعاتو
وقرأت عميو كثيراً وسمعت...ال يألؼ ألحد غيره وربما جاءني إلى منزلي وأسمعني وأسمع
"...أىمي وأوالدي(
29
)
، وأشار إلى أخذ القراآت عمى ابف العسقالني (ت793
)ىػ(
30
)
، عندما
رحؿ إليو مع أخيو قائالً: "... رحمت بأخيو لقراءة القراآت عمى ابف العسقالني... 1
-
:ثقافته قرأ القراآت عمى ابف الجزري(
42
)
، ونور
الديف أبو الحسف األشموني الذي اخذ عف ابف الجزري القراآت
كما يقوؿ ابف العماد(
43
)
،
وبدر الديف محمد بف أبي بكر ألمشدي "...وسمع المسند عمى الخير الممتوني وابف
"...الجزري(
44
)
، وعمي بف محمد الجعبري الذي درس مصنفاتو عمى ابف الجزري كما يقوؿ
ألنعيمي "...قدـ دمشؽ سنة اثنتيف وثالثيف واخذ عنو شياب الديف ألطيبي الحديث ومصنف ات
"...ابف الجزري(
45
) ا
ر
كانت الرحمة في طمب العمـ مف لوازـ طريؽ العمماء ومنيجيـ في التحصيؿ العممي ،
إذ كاف طالب العمـ يأخذ مف شيوخ بمده ثـ يرحؿ إلى البمداف األخرى لألخذ مف عممائيا
ًواالستفادة منيـ قدر اإلمكاف ، ويشير لنا ابف الصالح بيذا الخصوص قائال : "...وا ذا فرغ مف
"...سماع العوالي والميمات التي ببمده فميرحؿ إلى غيره(
46
)
، وبما إف ابف الجزري نيؿ العمـ
وىو صغير فكانت أولى رحالتو العممية مع والده وأخيو ولـ يحح وجيتو كما يقوؿ: "...ولما
"...رحمت بأخيو لقراءة القراآت... قرأ معو القرآف بالقراآت(
47
)
، أما ر حمتو الثانية التي لـ )
987
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
يحدد وجيتيا كما يقوؿ "... قرأ فييا القراآت العشر والشاطبية عمى إبراىيـ بف احمد
"...الشامي(
48
)
، ثـ رحؿ إلى بالد الروـ رسوالً فكانت الوقعة التيمورية ، كما يقوؿ "... لما
وقعت الفتنة التيمورية بالروـ كاف معي عندما طمبني األمير تيمورلنؾ فأ ًرسمتو عني رسوال
"...إلى السمطاف الناصر فرج بف برقوؽ(
49
)
..." ، وكانت لو رحمة أخرى لمحج ، كما يقوؿ
"...توجيت إلى الحج وجاورت(
50
)
5
-
:تقمدها
الوظائف الت جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
يحدد وجيتيا كما يقوؿ "... قرأ فييا القراآت العشر والشاطبية عمى إبراىيـ بف احمد
"...الشامي(
48
)
، ثـ رحؿ إلى بالد الروـ رسوالً فكانت الوقعة التيمورية ، كما يقوؿ "... 1
-
:ثقافته لما
وقعت الفتنة التيمورية بالروـ كاف معي عندما طمبني األمير تيمورلنؾ فأ ًرسمتو عني رسوال
"...إلى السمطاف الناصر فرج بف برقوؽ(
49
)
..." ، وكانت لو رحمة أخرى لمحج ، كما يقوؿ
"...توجيت إلى الحج وجاورت(
50
)
5تقم ها
ال ظائف الت ( العدد2
( ) اجمللد91
)
حزيران
2222 جملة جامعة االنبار للعلوم االنسانية تصدر ابف الجزري لإلقراء والتدريس واإلمالء في المدارس المنتشرة آنذاؾ، وأقبؿ
عميو طمبة العمـ ، لذا كانت أولى وظائفو تقمده وظائؼ أخيو بعد وفاتو في مصر بأمر مف
السمطاف أشرؼ برسباي ، كما يقوؿ "...وواله السمطاف األشرؼ برسباي وظائؼ أخيو أبي
الفتح رحمو اهلل التي كاف أخذىا عمى مشيخة اإلقراء بالمدرسة العادلية الكبرى والمشيخة
الكبرى بمدرسة أـ صالح وتدريس الصالحية بدم شؽ والتصدير بالجامع األموي وتدريس
"...األتابكية بسفح قاسيوف(
51
) ، ويذكر الحسني انو ولي قضاء شيراز(
52
)
، وعيف لقضاء بالد
الشاـ(
53
)
، ومف جممة اىتمامو بالقراء أنشأ ليـ مدرسة خاصة ودرس فييا كما يقوؿ ابف
..العماد "...وعمر لمقراء مدرسة سماىا دار القرآف وأقرأ الناس". (
54
). 6
-
:مؤلفاته كاف البف الجزري عدداً مف المؤلفات التي جعمت منو مؤلفاً يشار لو بالبناف ، وكاف
السائد منيا جاء في القراآت متطابقاً وميولو إذ كاف مقرئا قبؿ كؿ شيء ، ل ذا كانت مؤلفاتو
عمى النحو اآلتي))، منيا عمى سبيؿ ((ممخص تاريخ اإلسالـ(
55
) ، ((ذات الشفاء ف ي سيرة
))النبي والخمفاء(
56
)
))، ((فضائؿ القرآف(
57
)
))، ((سالح المؤمنيف(
58
)
))، ((منجد المقرئيف(
59
)
،
((الحصف الحصيف في األدعية واألذكار المأثورة)) ، وحاشية سماىا ((مفتاح الحصف
))الحصيف(
60
)
))، ((التتمة في القراآت(
61
)
))، ((تحبير النشر في القراآت العشر(
62
)
، ((الدرة
))المضيئة في القراآت(
63
)
، ((المقدمة الجزرية–
))أرجوزة في التجويد(
64
)
،((أسنى المطالب
))في مناقب عمي ابف أبي طالب (رضي اهلل عنو(
65
)
، ((اليداية في عمـ الرواية في
))المصطمح(
66
)
))، ((الزىر الفائح في ذكر مف نثره عف الذنوب والقبائح(
67
)
، ((شرح الجزرية
))في عمـ التجويد المسمى الحواشي المفيمة في شرح المقدمة(
68
)
، ((عدة الحصف الحصيف )
988
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
))مف كالـ سيد المرسميف(
69
)
))، ((المصعد األحمد في ختـ مسند اإلماـ احمد(
70
)
، ((المقدمة
))في تجويد القرآف(
71
)
))، ((منجد المقرئيف ومرشد الطالبيف(
72
). المبحث الثاني : الوظيفة والمهنة في كتاب غاية النهاية
أوال: منهج ابن الجزري في كتاب غاية النهاية في طبقات القراء
إف تنوع اتجاىات األعالـ المترجـ ليـ مف جية ،وكثرة الموارد التي اعتمدىا ،ومدى
توافرىا وتبايف نوعيتيا مف جية أخرى ،أدت إلى التبايف في المادة التي احتوتيا كؿ
ترجمة،ولعؿ اليدؼ الذي توخاه ابف الجزري في ترجمتو ليؤالء األعالـ لمتنويو بيـ وبمكانتيـ
ومشاركاتيـ في الثقافة العربية اإلسالمية أوال،وفي الحياة العامة ثانيا ،ألنو ترجـ لمختمؼ
شرائح القراء في المجتمع اإلسالمي آنذاؾ،مف خالؿ رفدىا بالجديد ة ،كال مف زاوية اىتمامو
يجعمنا نمتمس ق سما مف السمات العامة في الترجمة الواحدة ،عمى إننا نالحظ في عدد مف
تراجمو الطويمة تنظيما واضحا،لذا تضمف الكتاب مجموعة كبيرة مف التراجـ ولـ يقتصر
عمى عدد مف المشيوريف ،كما انو لـ يقتصر في تراجمو عمى أعياف بمد معيف مف القراء بؿ
شممت تراجمو أعالـ القراء لمعظ.ـ البالد اإلسالمية ( العدد2
( ) اجمللد91
)
حزيران
2222 المبحث الثاني : الوظيفة والمهنة في كتاب غاية النهاية
أوال: منهج ابن الجزري في كتاب غاية النهاية في طبقات القراء أوال: منهج ابن الجزري في كتاب غاية النهاية في طبقات القراء
إف تنوع اتجاىات األعالـ المترجـ ليـ مف جية ،وكثرة الموارد التي اعتمدىا ،ومدى
توافرىا وتبايف نوعيتيا مف جية أخرى ،أدت إلى التبايف في المادة التي احتوتيا كؿ
ترجمة،ولعؿ اليدؼ الذي توخاه ابف الجزري في ترجمتو ليؤالء األعالـ لمتنويو بيـ وبمكانتيـ
ومشاركاتيـ في الثقافة العربية اإلسالمية أوال،وفي الحياة العامة ثانيا ،ألنو ترجـ لمختمؼ
شرائح القراء في المجتمع اإلسالمي آنذاؾ،مف خالؿ رفدىا بالجديد ة ،كال مف زاوية اىتمامو
يجعمنا نمتمس ق سما مف السمات العامة في الترجمة الواحدة ،عمى إننا نالحظ في عدد مف
تراجمو الطويمة تنظيما واضحا،لذا تضمف الكتاب مجموعة كبيرة مف التراجـ ولـ يقتصر
عمى عدد مف المشيوريف ،كما انو لـ يقتصر في تراجمو عمى أعياف بمد معيف مف القراء بؿ
شممت تراجمو أعالـ القراء لمعظ.ـ البالد اإلسالمية ااإا
ونالحظ إف المادة الموجودة في كؿ ترجمة تختمؼ عف األخرى حسب طبيعة المترجـ
لو مف القراء وقيمتو العممية أو األدبية ،ومكانتو السياسية مف جية أخرى،وعدد الموارد التي
اعتمدىا ابف الجزري ونوعيتيا مف جية أخرى،ومف السيؿ إننا نجد اختالفا واضحا بيف
ترجمة
مقرئ عف أخر مثال،أو ترجمة المحدث،أو الفقيو ،أو الصوفي،وغيرىـ،إذ نجد أحيانا إف بعض
التراجـ التبمغ إال كممتيف أو كممة واحدة ،مثؿ ذلؾ ترجمة محمد بف برطاؿ(( شيخ
))البمقيني(
73
)
،وترجمة يحيى بف احمد بف سميماف أبو زكريا الجذامي األندلسي الذي قاؿ
عنو(( يعرؼ
))بابف موريف(
74
)
، وأحيانا تقتصر الترجمة عمى ذكر االسـ فقط،كما جاء في
))ترجمة (( موسى بف عبد الرحمف البيروني أبو عمراف الصباغ(
75
)
،وأحيانا التتعدى الترجمة
اسـ المترجـ لو والمصدر الذي اخذ منو ترجمتو ،كما في ترجمة احمد بف عبد اهلل بف
محمد بف عبد اهلل بف ىارو ف الصيدالني الوراؽ قائال(( كذا وقع في غاية أبي العالء وىو
))احمد بف محمد بف عبد اهلل بف ىاروف(
76
)
،أو يشير فقط إلى اسمو واحد مؤلفاتو فقط،كما
))في ترجمة عمي بف محمد أبو الحسف الفارسي((مؤلؼ كتاب عمؿ الغاية(
77
)
،أو انو قرأ عمى
احد األشخاص فقط،كما في ترجمة محمد بف محمد بف خميؿ أبو بكر السكوني((قرأ عميو أبو )
989
) ( العدد2
( ) اجمللد91
)
حزيران
2222 جملة جامعة االنبار للعلوم االنسانية ))البركات البمفيقي(
78
)
، أو قرأ عميو احد فقط،كما في ترجمة محمد بف عبد اهلل بف وىب أبو
))بكر القضاعي ،مقرئ((قرأ عميو محمد بف عبد الرحمف بف سيؿ(
79
)،
ويحدد أيضا المدة
المقرونة ببقاء مترجمو عند تمقي القراءة عمى يده،كم ا في ترجمة محمد بف عبد اهلل بف محمد
البغدادي
((يعرؼ بابف عبد المقرئ،روى القراءات والشاطبية ببغداد عف محمد بف يعقوب بف
بدراف الجرائدي ،قرأ عميو محمد بف محمود بف السمرقندي ،بقي إلى بعد األربعيف وسبعمائة
))واهلل اعمـ(
80
)
،وترجع األسباب في تغير حجـ بعض التراجـ إلى كثرة المعمومات المتوافرة لديو
عف المترجـ لو مف عدميا فقد شكؿ الكـ اليائؿ لممعمومات عف عدد المترجـ ليـ ،وأحيانا تبمغ
الترجمة مائة وواحد وعشروف سطرا في خمس صفحات
(( اواقؿ،كما ىو الحاؿ في ترجمة عمي
))بف حمزة بف عبد اهلل بف بيمف بف فيروز االسدي موالىـ(
81
) ،
أو تصؿ في بعض األحياف
إلى أربع صفحات كترجمة المؤلؼ لنفسو عمى عادة المحدثيف(
82
،)
وغيرىا مف التراجـ
،وبصوره عامة فاف اإلسياب الذي حصؿ في بعض تراجمو ىو في حقيقة األمر نتيجة
طبيعية خاصة النجازات المترجـ ليـ مف القراء الفكرية والعممية ومشاركاتيـ الحضارية في
مجاال ت الحية المختمفة ،وما ورد عنيـ مف حكايات وجرح وتعديؿ تعد واحدة مف العوامؿ التي
.نتجت في جعؿ بعض التراجـ طويمة غير إف السمة العامة لتراجـ الكتاب ىي اإليجاز قياسا إلى كتب التراجـ
األخرى،ولعمو يقصد مف ذلؾ العناية الخاصة بذكر األمور الميمة مف غير تفصيؿ كبير ف ي
األمور األخرى،كما أشار إلى ذلؾ في كتابو قائال.."(( فيذا كتاب "غاية النياية . ( العدد2
( ) اجمللد91
)
حزيران
2222 ارجوا أف
يجمع بيف الرواية والدراية اختصري فيو
...كتاب طبقات القراء الكبير))
(
83
)
،وىو خير مايدؿ
عمى ذلؾ إننا نستطيع أف نميز المنيج العاـ الذي اختطو ابف الجزري لنفسو الذي اتضح فيو
،أسموبو في ترتيب الترجمة،وذكر محتوياتيا مف العمماء
،والقراء، واألدباء، والمحدثيف
،والصوفية، ونحوىـ
:باألمور اآلتية ( العدد2
( ) اجمللد91
)
حزيران
2222 ( العدد2
( ) اجمللد91
)
حزيران
2222 جملة جامعة االنبار للعلوم االنسانية ))مف كالـ سيد المرسميف(
69
)
))، ((المصعد األحمد في ختـ مسند اإلماـ احمد(
70
)
، ((المقدمة
))في تجويد القرآف(
71
)
))، ((منجد المقرئيف ومرشد الطالبيف(
72
). ثانيا-
.الوظيفة والمهنة عني ابف الجزري في كتابو ((غاية النياية)) بذكر الوظائؼ التي شغميا المترجـ
ليـ،وتظير براعتو في ىذا المجاؿ مف خالؿ تتبعو الوظائؼ التي ينو ذبيا صاحب الترجمة
وتدرجو في المناصب محددا مكاف الوظيفة سواء كاف في محمة أو مدينة أو بمدة وغيرىا،نحو
قولو في ترجمة إسماعيؿ بف م..((حمد بف عبد اهلل التستري . إماـ صفة صالح الديف )
990
) ( العدد2
( ) اجمللد91
)
حزيران
2222 جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
..بالصالحية ثـ خانقاه سرياقوس
. وكاف شي
خ القراءات بالمدرس...ة الفاضمية))
(
84
)،
وا سماعيؿ
ب..ف محمد بف عمي بف عبد اهلل((... قاضي القضاة
. ثـ قدـ حماة فأ قاـ بيا وولي قضاء
...المالكية))
(
85
)
..((،ومحمد بف محمد بف إبراىيـ
. قاضي الج..ماعة بغرناطة . وقمد قضاء
المرية...مدة ثـ نقؿ إلى قضاء غرناطة))
(
86
)،أو يقتصر في ىذا المجاؿ عم ى ذكر منصب
صاحب الترجمة...(( في مدينة واحدة،نحو قولو
...قاضي القضاة بدمشؽ))
(
87
)
،وبصورة
عامة يكوف قد وضح لنا بعض الجوانب اإلدارية في عدد مف المدف العربية واإلسالمية،كما
عني بذكر وظيفة المترجـ لو الرئيسة التي تعد مف األمور الميمة في التراجـ،نحو قولو في
ترجمة احمد بف عبد الرحمف بف محمد بف سعيد القرطبي
..((... قاضي الجماعة)) . (
88
)
،
واحمد بف مح...مد بف عبدوف((... القاضي))
(
89
)،
ويالحظ في ىذا انو دأب عمى ذكر بعض
المدارس التي درس بيا المترجـ لو والسيما مدارس القاىرة المختمفة(
90
). لذا يعد كتاب ((غاية النياية)) في مقدمة
المصادر التي يمكف االعتماد عمييا في
.دراسة الحركة الفكرية في القرف التاسع اليجري/ الخامس عشر الميالدي
وغالبا كاف يشير إلى تولي مترجمو مينتو مع ذكر سنة ومكاف توليو الوظيفة أو
المينة وما يرافقيا مف أحداث دالة عمى ذلؾ مف حيث تولي الحكـ في تمؾ الحقبة وىذا دلي ؿ
عمى دقتو وحرصو إيراد روايتو مضبوطة وىذا جزء مف منيجو في رواياتو،نحو قولو في
ترجمة إبراىيـ
..((بف عبد اهلل بف عمي ألحكري . شيخ مشايخ اإلقراء بالديار
))المصرية(
91
)
..((،واحمد بف عبد اهلل
. عيف لقضاء مصر سنة ثالث وث الثيف وخمسمائة أياـ
. .العبيديف)) . (
92
)
،وشر
يح ب..((ف محمد بف شريح الرعيني. .ولي خطابة اشبيمية وقضاءىا
.. ))
(
93
)، وا سماع.((يؿ بف محمود بف احمد الدمشقي
.. خطيب الجامع العتيؽ بالمحمة الك برى
..مف ديار مصر)) . (
94
)
..،وعمي بف عبد الكافي((... قاضي دمشؽ
. وقدـ دمشؽ قا ضيا سنة
..تسع وثالثيف وسبعمائة)). ثانيا-
.الوظيفة والمهنة (
95
) ،أو يشير إلى
تولي مترجمو لوظيفة القاضي مف غير ذكراية
معمومات أخرى عنو ،نحو قولو في ترجمة خالد بف يزيد
...((... قاضي البمقاء))
(
96
)
،أو مف
كاف يتولى وظيفتو وكالة ،نحو قولو في ترجمة عبد اهلل بف محمد السامر ي ((. . . الوكيؿ عند
..القضاة)) . (
97
)
،وكاف يشير إلى تولي مترجمو لمينتو أو وظيفتو ألوؿ مرة سواء استمر فييا
أو تـ عزلو منيا ذاكرا أسباب ذلؾ،نحو قولو في ترجم..((ة عبد السالـ بف عمي بف عمر . وىو أوؿ مف ولي قضاء المالكية بدمشؽ لما صارت القضاة عمى أربعو عمى كره منو فباشر جملة جامعة االنبار للعلوم االنسانية ..بالصالحية ثـ خانقاه سرياقوس
. وكاف شي
خ القراءات بالمدرس...ة الفاضمية))
(
84
)،
وا سماعيؿ
ب..ف محمد بف عمي بف عبد اهلل((... قاضي القضاة
. ثـ قدـ حماة فأ قاـ بيا وولي قضاء
...المالكية))
(
85
)
..((،ومحمد بف محمد بف إبراىيـ
. قاضي الج..ماعة بغرناطة . وقمد قضاء
المرية...مدة ثـ نقؿ إلى قضاء غرناطة))
(
86
)،أو يقتصر في ىذا المجاؿ عم ى ذكر منصب
صاحب الترجمة...(( في مدينة واحدة،نحو قولو
...قاضي القضاة بدمشؽ))
(
87
)
،وبصورة
عامة يكوف قد وضح لنا بعض الجوانب اإلدارية في عدد مف المدف العربية واإلسالمية،كما
عني بذكر وظيفة المترجـ لو الرئيسة التي تعد مف األمور الميمة في التراجـ،نحو قولو في
ترجمة احمد بف عبد الرحمف بف محمد بف سعيد القرطبي
..((... قاضي الجماعة)) . (
88
)
،
واحمد بف مح...مد بف عبدوف((... القاضي))
(
89
)،
ويالحظ في ىذا انو دأب عمى ذكر بعض
المدارس التي درس بيا المترجـ لو والسيما مدارس القاىرة المختمفة(
90
). ا
.دراسة الحركة الفكرية في القرف التاسع اليجري/ الخامس عشر الميالدي
وغالبا كاف يشير إلى تولي مترجمو مينتو مع ذكر سنة ومكاف توليو الوظيفة أو
المينة وما يرافقيا مف أحداث دالة عمى ذلؾ مف حيث تولي الحكـ في تمؾ الحقبة وىذا دلي ؿ
عمى دقتو وحرصو إيراد روايتو مضبوطة وىذا جزء مف منيجو في رواياتو،نحو قولو في
ترجمة إبراىيـ
..((بف عبد اهلل بف عمي ألحكري . شيخ مشايخ اإلقراء بالديار
))المصرية(
91
)
..((،واحمد بف عبد اهلل
. عيف لقضاء مصر سنة ثالث وث الثيف وخمسمائة أياـ
. .العبيديف)) . (
92
)
،وشر
يح ب..((ف محمد بف شريح الرعيني. .ولي خطابة اشبيمية وقضاءىا
.. ))
(
93
)، وا سماع.((يؿ بف محمود بف احمد الدمشقي
.. خطيب الجامع العتيؽ بالمحمة الك برى
..مف ديار مصر)) . (
94
)
..،وعمي بف عبد الكافي((... قاضي دمشؽ
. ثانيا-
.الوظيفة والمهنة وقدـ دمشؽ قا ضيا سنة
..تسع وثالثيف وسبعمائة)). (
95
) ،أو يشير إلى
تولي مترجمو لوظيفة القاضي مف غير ذكراية
معمومات أخرى عنو ،نحو قولو في ترجمة خالد بف يزيد
...((... قاضي البمقاء))
(
96
)
،أو مف
كاف يتولى وظيفتو وكالة ،نحو قولو في ترجمة عبد اهلل بف محمد السامر ي ((. . . الوكيؿ عند
..القضاة)) . (
97
)
،وكاف يشير إلى تولي مترجمو لمينتو أو وظيفتو ألوؿ مرة سواء استمر فييا
أو تـ عزلو منيا ذاكرا أسباب ذلؾ،نحو قولو في ترجم..((ة عبد السالـ بف عمي بف عمر . وىو أوؿ مف ولي قضاء المالكية بدمشؽ لما صارت القضاة عمى أربعو عمى كره منو فباشر )
991
) ( العدد2
( ) اجمللد91
)
حزيران
2222 جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
تسع سنيف فمما مات رفيقو
...الحنفي ابف عطاء عزؿ نفسو))
(
98
)
،وعمي بف محمد ((قاضي
دمشؽ أ...وؿ مف ولي دمشؽ مف دمشؽ مطمقا))
(
99
)،
..((ومحمد بف عمي بف صالح . ولي
إمامة المدرسة الصرغتمش.. ية
. وباشر القضا.ء في قناطر السباع ظاىر القاىرة)) . (
100
). وبصورة عامو نجد انو وضح لنا مف خالؿ تراجمو بعض جوانب الحالة اإلدارية لعدد
مف ال مدف العربية اإلسالمية وىذا االتجاه المنيجي أظيره لنا مف خالؿ التعامؿ مع ىذا
الجانب الياـ مف التراجـ،فيما إذا كاف المترجـ لو مف المعدليف،وذكر لفظ المعدؿ(
101
)
،نحو
قولو في ترجمة..احمد بف عبد القادر بف رافع((... مف عدوؿ إسكندرية)) . (
102
)
،أو متصدر
المينة،نحو قولو
في ترجمة احمد بف عبد اهلل بف محمد البغدادي
((... متصدر
...عدؿ))
(
103
)،
ويعود ىذا إلى أىمية ىذا األمر في توثيؽ المترجـ لو وقبولو في المناصب
ًالدينية والسيما القضاء زيادة عمى ذلؾ فيو يذكر فيما إذا كاف المترجـ لو شروطيا(
104
)، جملة جامعة االنبار للعلوم االنسانية جملة جامعة االنبار للعلوم االنسانية
حزيران
2222
تسع سنيف فمما مات رفيقو
...الحنفي ابف عطاء عزؿ نفسو))
(
98
)
،وعمي بف محمد ((قاضي
دمشؽ أ...وؿ مف ولي دمشؽ مف دمشؽ مطمقا))
(
99
)،
..((ومحمد بف عمي بف صالح . ولي
إمامة المدرسة الصرغتمش.. ية
. وباشر القضا.ء في قناطر السباع ظاىر القاىرة)) . (
100
). ثانيا-
.الوظيفة والمهنة وبصورة عامو نجد انو وضح لنا مف خالؿ تراجمو بعض جوانب الحالة اإلدارية لعدد
مف ال مدف العربية اإلسالمية وىذا االتجاه المنيجي أظيره لنا مف خالؿ التعامؿ مع ىذا
الجانب الياـ مف التراجـ،فيما إذا كاف المترجـ لو مف المعدليف،وذكر لفظ المعدؿ(
101
)
،نحو
قولو في ترجمة..احمد بف عبد القادر بف رافع((... مف عدوؿ إسكندرية)) . (
102
)
،أو متصدر
المينة،نحو قولو
في ترجمة احمد بف عبد اهلل بف محمد البغدادي
((... متصدر
...عدؿ))
(
103
)،
ويعود ىذا إلى أىمية ىذا األمر في توثيؽ المترجـ لو وقبولو في المناصب
ًالدينية والسيما القضاء زيادة عمى ذلؾ فيو يذكر فيما إذا كاف المترجـ لو شروطيا(
104
)،
أو احد الشيود(
105
)، او
،عكس ذلؾ نحو
..((قولو في ترجمة احمد بف حرب . وليس
ىذا
..بالمعدؿ الذي ىو احمد بف حرب
. وليس أيضا بالمعدؿ الذي قرأ عمى محمد بف
..وىب)). (
106
)،ويشير إلى تسمسؿ ىذه المفظة في نسب مترجمو وشيرتو فييا ،نحو قولو ف ي
..ترجمة احمد بف حرب بف غيالف((... أبو جعفر المعدؿ)) . (
107
)،وحرص أيض ا عمى ذكر
تولي مترجمو أكثر مف وظيفة في أماكف مختمفة ،نحو قولو في ترجمة
الحسيف بف سميماف بف
..((فزارة . ولي تدريس الطرخانية ومشيختو الزنجيميو ثـ مشيخة...ألمقدميو))
(
108
)،
واحمد بف
محمد..بف احمد((... المعدؿ العطار)) . (
109
)،
واحم د بف عبدوف((الصيدالني
...القاضي))
(
110
)،
..و((... الصفار... المعدؿ)) . (
111
)
..،و((البزاز السمسار)) . (
112
)،
.. و((.. . الطبيب الكحاؿ)) . (
113
)
.أو نراه يحاوؿ تقديـ نوع مف االختصار عندما يقتصر
عمى ذكر مف ولي مف مت،رجميو لمينة واحدة فقط ومثاؿ ذلؾ
: البزاز(
114
)، االسكاؼ(
115
)،
الصفار(
116
)،العطاردي(
117
). ولـ يغفؿ اب ف الجزري في اإلشارة إلى شيرة مترجمو نسبة إلى مينتو ميما كانت سواء
ا
القا
ة ا
ق ل ف
ا
فة
م ة أ إ ا ة أ
ف (( ـا
أو احد الشيود(
105
)، او
،عكس ذلؾ نحو
..((قولو في ترجمة احمد بف حرب . وليس
ىذا
..بالمعدؿ الذي ىو احمد بف حرب
. وليس أيضا بالمعدؿ الذي قرأ عمى محمد بف
..وىب)). (
106
)،ويشير إلى تسمسؿ ىذه المفظة في نسب مترجمو وشيرتو فييا ،نحو قولو ف ي
..ترجمة احمد بف حرب بف غيالف((... أبو جعفر المعدؿ)) . (
107
)،وحرص أيض ا عمى ذكر
تولي مترجمو أكثر مف وظيفة في أماكف مختمفة ،نحو قولو في ترجمة
الحسيف بف سميماف بف
..((فزارة . ثانيا-
.الوظيفة والمهنة (
130
)،
وغيرىا مف الميف
والوظائؼ المتنوعة التي زاوليا أو مارسيا البعض مف تراجمو، منيا عمى سبيؿ المثاؿ ال
الحصر :العطار(
131
)،العشاب(
132
)، الدالؿ(
133
)،االسكافي(
134
)
.،وغيرىا
وأشار أيضا إلى المناصب الكبار التي توالىا مترجميو، وىذا األمر يعطي
انطباع عمى نوع التدرج اإلداري الذي ك
اف سائدا آنذاؾ ألىمية ىذا األمر
في توثيؽ المترجـ
لو وقبولو تمؾ المناصب ،نحو قولو في ترجمة احمد بف احمد اليكار ي((... تولي المناصب
..الكبار)) . (
135
)،ومح..مد بف الحسف((... وليس معدؿ)) . (
136
). ا
أ
ل
ما
لا
أ
أ جملة جامعة االنبار للعلوم االنسانية وكجزء مف اىتمامو بمينة مترجمو فأنو يشير إلى شيرتو نسبة إلى مينتو ميما كاف
نوعيا ،نحو قولو في ترجمة
..إبراىيـ بف إسماعيؿ المصري((... مشيور عدؿ)) . (
120
)
،أو
معروؼ نحو قولو في ترجمة إبرا ىيـ بف منصور بف عبد الصمد ((... المعروؼ
..باألدمي))
(
121
)،
..واحمد بف عثماف بف العباس((... الوراؽ ورّاؽ خمؼ مشيور . ))
(
122
)،ومحمد بف
..سعيد بف عمي((الشيير بالطراز)) . (
123
). ))ي(())
وعندما ينقؿ روايتو في بعض األحياف عف مترجمو مينتو لمتعريؼ بو أو لتميزه عف
آخر إذا تشابو باالسـ معو وغير ذلؾ مف األسباب،نحو قولو في ترج
مة إبراىيـ ب ف احمد
..((االربمي . روى القراءة عف احمد بف الحسف بف..عمي المعدؿ)) . (
124
)
،وا براىيـ
..((السمسار
. روى القراءة عرضا عف احمد بف عمي ا...لبزار فيما قالو النقاش وغيره))
(
125
). وكاف مف منيجو العاـ في التراجـ اإلشارة إلى وظيفة مترجمو عند إيراد اسـ مترجمو
عمى النحو
التالي: قولو(( إبراىيـ بف الحسف بف إبراىيـ بف يحيى بف عبد ال رحمف أبو اسحؽ
...األشعري النقاش))
(
126
)،و((إبراىي..ـ بف الخضر بف إبراىيـ النقاش)) . (
127
)،
و((إبراىيـ بف
..عمي الحداد)). (
128
)،و((احم..د بف إسحاؽ بف إبراىيـ الخياط)) . (
129
)،
و((احمد بف محمد
بف حسف أبو العبا
س
.بف الغماز األنصاري قاضي تونس)) .. (
130
)،
وغيرىا مف الميف
والوظائؼ المتنوعة التي زاوليا أو مارسيا البعض مف تراجمو، منيا عمى سبيؿ المثاؿ ال
الحصر :العطار(
131
)،العشاب(
132
)، الدالؿ(
133
)،االسكافي(
134
)
.،وغيرىا اا
وأشار أيضا إلى المناصب الكبار التي توالىا مترجميو، وىذا األمر يعطي
انطباع عمى نوع التدرج اإلداري الذي ك
اف سائدا آنذاؾ ألىمية ىذا األمر
في توثيؽ المترجـ
لو وقبولو تمؾ المناصب ،نحو قولو في ترجمة احمد بف احمد اليكار ي((... تولي المناصب
..الكبار)) . (
135
)،ومح..مد بف الحسف((... وليس معدؿ)) . (
136
). ثانيا-
.الوظيفة والمهنة ولي تدريس الطرخانية ومشيختو الزنجيميو ثـ مشيخة...ألمقدميو))
(
108
)،
واحمد بف
محمد..بف احمد((... المعدؿ العطار)) . (
109
)،
واحم د بف عبدوف((الصيدالني
...القاضي))
(
110
)،
..و((... الصفار... المعدؿ)) . (
111
)
..،و((البزاز السمسار)) . (
112
)،
.. و((.. . الطبيب الكحاؿ)) . (
113
)
.أو نراه يحاوؿ تقديـ نوع مف االختصار عندما يقتصر
عمى ذكر مف ولي مف مت،رجميو لمينة واحدة فقط ومثاؿ ذلؾ
: البزاز(
114
)، االسكاؼ(
115
)،
الصفار(
116
)،العطاردي(
117
). ولـ يغفؿ اب ف الجزري في اإلشارة إلى شيرة مترجمو نسبة إلى مينتو ميما كانت سواء
عممية أو إدارية أو حرفة وغيرىا ،نحو قولو في ترجمة احمد بف عبد القادر بف را فع((... مف
..عدوؿ اإلسكندرية)) . (
118
)
،أو متصدر مينة في مدينة ،نحو قولو في ترجمة احمد بف عبد
اهلل بف مح...((مد البغدادي
..متصدر عدؿ)) . (
119
). )
992
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
وكجزء مف اىتمامو بمينة مترجمو فأنو يشير إلى شيرتو نسبة إلى مينتو ميما كاف
نوعيا ،نحو قولو في ترجمة
..إبراىيـ بف إسماعيؿ المصري((... مشيور عدؿ)) . (
120
)
،أو
معروؼ نحو قولو في ترجمة إبرا ىيـ بف منصور بف عبد الصمد ((... المعروؼ
..باألدمي))
(
121
)،
..واحمد بف عثماف بف العباس((... الوراؽ ورّاؽ خمؼ مشيور . ))
(
122
)،ومحمد بف
..سعيد بف عمي((الشيير بالطراز)) . (
123
). وعندما ينقؿ روايتو في بعض األحياف عف مترجمو مينتو لمتعريؼ بو أو لتميزه عف
آخر إذا تشابو باالسـ معو وغير ذلؾ مف األسباب،نحو قولو في ترج
مة إبراىيـ ب ف احمد
..((االربمي . روى القراءة عف احمد بف الحسف بف..عمي المعدؿ)) . (
124
)
،وا براىيـ
..((السمسار
. روى القراءة عرضا عف احمد بف عمي ا...لبزار فيما قالو النقاش وغيره))
(
125
). وكاف مف منيجو العاـ في التراجـ اإلشارة إلى وظيفة مترجمو عند إيراد اسـ مترجمو
عمى النحو
التالي: قولو(( إبراىيـ بف الحسف بف إبراىيـ بف يحيى بف عبد ال رحمف أبو اسحؽ
...األشعري النقاش))
(
126
)،و((إبراىي..ـ بف الخضر بف إبراىيـ النقاش)) . (
127
)،
و((إبراىيـ بف
..عمي الحداد)). (
128
)،و((احم..د بف إسحاؽ بف إبراىيـ الخياط)) . (
129
)،
و((احمد بف محمد
بف حسف أبو العبا
س
.بف الغماز األنصاري قاضي تونس)) .. ثانيا-
.الوظيفة والمهنة (
147
)
،وكاف لو نوع مف الميوؿ التي اتجو بيا في إيراد مينة
أو وظيفة مترجمو مف ناحية التقديـ والتأخير في النسبة الى مكاف ما،فكاف يقدـ المينة عمى
أصؿ المترجـ لو،نحو قولو:
..(( .. الخياط الكوفي)) . (
148
)
. .،و((... الطناجيري البغدادي
. ))
(
149
)،
أو عكس ذل ...(( :ؾ،نحو قولو..الجزري المقصاتي. ))
(
150
)
..((،و. الكوفي
..المعدؿ)). (
151
)
..،و((الكوفي الصيرفي)) . (
152
)
. وإلظيار مدى التطور الذي كاف عميو العصر آنذاؾ فقد كاف يعرض في بعض
تراجمو اال..ختصاص الدقيؽ ليـ،نحو قولو((... الطبيب الكحاؿ)) . (
153
)
..((،و . النقاش
لمخواتـ ويقاؿ الثقا
ب القصاص أي..ضا حمدوية المؤلؤي)) . (
154
)
،ومف الحاالت التي كاف
هليحاوؿ فييا أبراز مينة مترجمو قبؿ ذكر اسمو دوف أف يعمؿ سبب ذلؾ،نحو قولو:(( الطبيب جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
..((الصمد بف سمطاف
ا . ك. ف متقنا العربية رأسا في الطب)) . (
140
)،ومحمد بف إبراىي..((ـ . ي..حترؼ التجارة وكاف عدال مرضيا)) . (
141
)
..،و((... القاضي الخطيب)). (
142
)
..((، و . الص..يرفي الكوفي إماـ في القراءة. ))
(
143
)
..،و((... ولي خطابة اشبيمية وقضاءىا)) . (
144
)،
..و((... الحداد إماـ طرسوس)) . (
145
). ( العدد2
( ) اجمللد91
)
حزيران
2222 جملة جامعة االنبار للعلوم االنسانية ..((الصمد بف سمطاف
ا . ك. ف متقنا العربية رأسا في الطب)) . (
140
)،ومحمد بف إبراىي..((ـ . ي..حترؼ التجارة وكاف عدال مرضيا)) . (
141
)
..،و((... القاضي الخطيب)). (
142
)
..((، و . الص..يرفي الكوفي إماـ في القراءة. ))
(
143
)
..،و((... ولي خطابة اشبيمية وقضاءىا)) . (
144
)،
..و((... الحداد إماـ طرسوس)) . (
145
). أواف مترجمو قدـ إلى مدينة مف البالد اإلسالمية فتولى مينة أو وظيفة ما بيا ،نحو
قولو في ترجمة احمد..(( بف عمي بف الشيخ فخر الديف . و قدـ دمشؽ فولي تدريس
..القصاعيف)) . (
146
)
..((،وأبو بكر بف يوسؼ . وقدـ دمشؽ وكتب في شيود تحت الشاعات
وكانت لو مشاركة في..فنوف)) . (
147
)
،وكاف لو نوع مف الميوؿ التي اتجو بيا في إيراد مينة
أو وظيفة مترجمو مف ناحية التقديـ والتأخير في النسبة الى مكاف ما،فكاف يقدـ المينة عمى
أصؿ المترجـ لو،نحو قولو:
..(( .. الخياط الكوفي)) . (
148
)
. .،و((... الطناجيري البغدادي
. ))
(
149
)،
أو عكس ذل ...(( :ؾ،نحو قولو..الجزري المقصاتي. ))
(
150
)
..((،و. ثانيا-
.الوظيفة والمهنة وأورد أيضا فضال إلى مينتو االصميو القراءة مينة أخرى إال انو عندما يعرض
الترجمة ينكر تولي مترجمو ىذه الوظيفة وال نعرؼ سبب اتجاىو ىذا ،نحو قولو في ترجمة
..((احمد بف حرب بف غيالف
. وكاف ثقة يعد مف الق،راء أيضا
وليس بالمعدؿ
...أيضا))
(
137
)
،،أو عكس ذلؾ انو برع في القراءة ومينتو األخرى نحػػػػ
ػػػو
قولو في ترجمة
..((كماؿ بف عمر التبريزي
. فاضؿ محقؽ برع
.. في القراءات والطب وغير ذلؾ)) . (
138
)،
..واحمد بف عمي بف محمد الشيخ((... وذكر لي انو أديب طبيب محدث)) . (
139
)
،وعبد وأشار أيضا إلى المناصب الكبار التي توالىا مترجميو، وىذا األمر يعطي
انطباع عمى نوع التدرج اإلداري الذي ك
اف سائدا آنذاؾ ألىمية ىذا األمر
في توثيؽ المترجـ
لو وقبولو تمؾ المناصب ،نحو قولو في ترجمة احمد بف احمد اليكار ي((... تولي المناصب
..الكبار)) . (
135
)،ومح..مد بف الحسف((... وليس معدؿ)) . (
136
). وأورد أيضا فضال إلى مينتو االصميو القراءة مينة أخرى إال انو عندما يعرض
الترجمة ينكر تولي مترجمو ىذه الوظيفة وال نعرؼ سبب اتجاىو ىذا ،نحو قولو في ترجمة
..((احمد بف حرب بف غيالف
. وكاف ثقة يعد مف الق،راء أيضا
وليس بالمعدؿ
...أيضا))
(
137
)
،،أو عكس ذلؾ انو برع في القراءة ومينتو األخرى نحػػػػ
ػػػو
قولو في ترجمة
..((كماؿ بف عمر التبريزي
. فاضؿ محقؽ برع
.. في القراءات والطب وغير ذلؾ)) . (
138
)،
..واحمد بف عمي بف محمد الشيخ((... وذكر لي انو أديب طبيب محدث)) . (
139
)
،وعبد )
993
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
..((الصمد بف سمطاف
ا . ك. ف متقنا العربية رأسا في الطب)) . (
140
)،ومحمد بف إبراىي..((ـ . ي..حترؼ التجارة وكاف عدال مرضيا)) . (
141
)
..،و((... القاضي الخطيب)). (
142
)
..((، و . الص..يرفي الكوفي إماـ في القراءة. ))
(
143
)
..،و((... ولي خطابة اشبيمية وقضاءىا)) . (
144
)،
..و((... الحداد إماـ طرسوس)) . (
145
). أواف مترجمو قدـ إلى مدينة مف البالد اإلسالمية فتولى مينة أو وظيفة ما بيا ،نحو
قولو في ترجمة احمد..(( بف عمي بف الشيخ فخر الديف . و قدـ دمشؽ فولي تدريس
..القصاعيف)) . (
146
)
..((،وأبو بكر بف يوسؼ . وقدـ دمشؽ وكتب في شيود تحت الشاعات
وكانت لو مشاركة في..فنوف)) . ثانيا-
.الوظيفة والمهنة ووضح لنا مف
خالؿ تراجمو بعض جوانب الحالة اإلدارية لعدد مف المدف العربية اإلسالمية وىذا االتجاه
المنيجي أظيره لنا مف خالؿ التعامؿ مع ىذا الجانب الياـ مف ال. تراجـ جملة جامعة االنبار للعلوم االنسانية ..((الغرناطي
. وعيف لمشيخة اإلقراء ب..المدرسة بغرناطة فامتنع تدينا)) . (
161
)
،أو عالقة
مترجمو مع شيوخو،نحو قولو في تر
جمة ع..(( بد الصمد بف احمد البغدادي . سمعت أبا بكر
المقصاتي يقوؿ طمب مني شيخنا عبد الصمد مقصا فعممتو وأثبتو بو ف ما أخذه حتى أعطاني
..فوؽ قيمتو)) . (
162
). النتائج اتبع منيجا قائما عمى أساس عرض وتحميؿ مالو
عالقة بوظيفة مترجمو ومينتو،
وىذا
المنيج عكس لنا اىتمامو العاـ بالتراجـ فقد حرص عمى إيرادىا بأدؽ تفاصيميا وأوسع
المعمومات عنيا،إذ عرضيا ضمف االسـ متبع في ذلؾ اتجاىات عدة وربطيا بشكؿ متداخؿ
مع المعمومات األخرى الخاصة بالترجمة سواء كانت موسعو أو مختصرة ،وجعميا مرتبطة
بشكؿ مباشر مع األحداث التي كانت متداخمة في التر جمة وعرضيا بأسموب إداري أو عممي
أو حرفي وغيرىا مف األحواؿ التي كانت عمييا البالد العربية واإلسالمية. ووضح لنا مف
خالؿ تراجمو بعض جوانب الحالة اإلدارية لعدد مف المدف العربية اإلسالمية وىذا االتجاه
المنيجي أظيره لنا مف خالؿ التعامؿ مع ىذا الجانب الياـ مف ال. تراجـ ثانيا-
.الوظيفة والمهنة الكوفي
..المعدؿ)). (
151
)
..،و((الكوفي الصيرفي)) . (
152
)
. وإلظيار مدى التطور الذي كاف عميو العصر آنذاؾ فقد كاف يعرض في بعض
تراجمو اال..ختصاص الدقيؽ ليـ،نحو قولو((... الطبيب الكحاؿ)) . (
153
)
..((،و . النقاش
لمخواتـ ويقاؿ الثقا
ب القصاص أي..ضا حمدوية المؤلؤي)) . (
154
)
،ومف الحاالت التي كاف
يحاوؿ فييا أبراز مينة مترجمو قبؿ ذكر اسمو دوف أف يعمؿ سبب ذلؾ،نحو قولو:(( الطبيب
بف
.. الحسف بف عبد اهلل بف حمداف)) . (
155
)،أو يذكر فقط اسـ مترجمو ومينتو ،نحو قولو:
))((ابف الكردي محمد بف يعقوب المعدؿ(
156
)
،أو
ذكر كنية مترجمو وم ينتو فقط((أبو طاىر
..الصيدالني)) . (
157
)
.،دوف أف يعمؿ سبب اتجاىو ىذا ،ونراه في بعض األحياف يحدد المدة التي تولى فييا مترجمو مينتو
نحو قولو في
ترجم..((ة العباس بف الفضؿ األنصاري
. قاضي ال..موصؿ . ولي القضاء بالموصؿ فانتقؿ
إلييا وأقاـ بيا قاضيا))إلى أف مات(
158
)
..((،وعبد الصمد بف حامد
. وكاف قد ولي ف ي آخر
..وقت قضاء القضاة بتبريز)) . (
159
)
،أو انو كاف يحدد تولي مترجمو لمينتو في أكثر مف
مكاف،نحو قولو في ترجمة.((محمد بف إبراىيـ أبو عبد اهلل. . مدرس الزنجيمية والبمخية
... ))
(
160
)،
وانو في بعض األحياف يشير إلى سيرة المترجـ لو مف خالؿ وظيفتو سواء قبولو
لتمؾ الوظيفة أو رفضيا لسبب أو آلخر،نحو قولو في ترجمة احمد بف عمي بف احمد )
994
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
..((الغرناطي
. وعيف لمشيخة اإلقراء ب..المدرسة بغرناطة فامتنع تدينا)) . (
161
)
،أو عالقة
مترجمو مع شيوخو،نحو قولو في تر
جمة ع..(( بد الصمد بف احمد البغدادي . سمعت أبا بكر
المقصاتي يقوؿ طمب مني شيخنا عبد الصمد مقصا فعممتو وأثبتو بو ف ما أخذه حتى أعطاني
..فوؽ قيمتو)) . (
162
). النتائج
اتبع منيجا قائما عمى أساس عرض وتحميؿ مالو
عالقة بوظيفة مترجمو ومينتو،
وىذا
المنيج عكس لنا اىتمامو العاـ بالتراجـ فقد حرص عمى إيرادىا بأدؽ تفاصيميا وأوسع
المعمومات عنيا،إذ عرضيا ضمف االسـ متبع في ذلؾ اتجاىات عدة وربطيا بشكؿ متداخؿ
مع المعمومات األخرى الخاصة بالترجمة سواء كانت موسعو أو مختصرة ،وجعميا مرتبطة
بشكؿ مباشر مع األحداث التي كانت متداخمة في التر جمة وعرضيا بأسموب إداري أو عممي
أو حرفي وغيرىا مف األحواؿ التي كانت عمييا البالد العربية واإلسالمية. االحاالت (
1
)
، غاية النياية في طبقات القراء ، عني بنشره براجستراسر ، (القاىرة ، مكتبة الخانجي1352
/ىػ
1933
، )ـ1
/
129
، ؛ شرح طيبة النشر في القراءات العشر ، ضبطو وراجعو الشيخ عمي محمد الضباع
ط1
، (مصطفى البابي الحمبي1950
ـ) ، ص ص3
-
4؛ شرح طيبة النشر في القراءات العشر
، حققو
وعمؽ عميو الشيخ أنس ميرة ، ط2
، (بيروت ، دار الكتب العممية1420
/ ىػ2000ـ) ، ص3
، أبو
المحاسف محمد بف عمي بف الحسيف بف حمزة الحسيني (ت765
ىػ) ، ذيؿ تذكرة الحفاظ ، تحقيؽ حساـ
، )الديف القدسي ، (بيروت ، دار الكتب العممية ، د.ت1
/
376
–
377؛ أبو الف ضؿ احمد بف عمي بف
محمد بف حجر العسقالني (ت852
ىػ) ، الدرر الكامنة في أعياف المائة الثامنة ، تحقيؽ د. االحاالت محمد عبد
المعيد خاف ، ط2
، (حيدر أباد اليند ، مجمس دائرة المعارؼ العثمانية1972
، )ـ1
/
33
؛ جماؿ الديف أبو
المحاسف يوسؼ بف تغري بردي االتابكي (ت874
، )ىػ ،النجوـ الزاىرة في مموؾ مصر والقاىرة ، (القاىرة
)المؤسسة المصرية العامة لمتأليؼ والترجمة والطباعة والنشر ، د.ت14
/
16
؛ شمس الديف محمد بف عبد
الرحمف السخاوي (ت902ىػ) ، التحفة المطيفة في تاريخ المدينة الشريفة ، ط1
(بيروت ، دار الكتب العممية
،
1933
، )ـ1
/
61
)، الضوء الالمع ألىؿ القرف التاسع ، (بيروت ، مكتبة دار الحياة ، د.ت2
/
193
؛ )
995
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 ( العدد2
( ) اجمللد91
)
حزيران
2222 ، ذيؿ طبقات الحفاظ لمذىبي ، نشر باعتناء وستفمد (غوتنجي1833
–
1834
، )ـ1
/
549
؛ عبد الرحمف
بف محمد أبو اليمف العميمي (ت927
ىػ) ، األنس الجميؿ بتاريخ القدس والخميؿ ، ط2
(النجؼ ، ، الحيدرية
1968
، )ـ2
/
454
؛ عبد القادر بف محمد ألنعيمي الدمشقي (ت927
، ىػ) ، الدارس في تاريخ المدارس
تحقيؽ إبراىيـ شمس الديف ، ط1
، (بيروت ، دار الكتب العممية1410
، )ىػ1
/
245
؛ عصاـ الديف أبو
الخير احمد بف مصطفى طاش كبرى زادة (ت968
ىػ) ، مفتاح السعادة ومصباح السيادة في موضوعات
، العموـ ، تحقيؽ كامؿ بكري وعبد الوىاب أبو النور ، (القاىرة ، دار الكتب الحديثة1968
، ) ـ1
/
392
؛
، والشقائؽ العمانية في عمماء الدولة العثمانية ، (بيروت ، دار الكتاب العربي1975
، )ـ1
/
39
؛ ابو
الحسنات محمد بف عبد الحي بف الحافظ محمد بف عبد الحميـ بف محمد بف أميف المكنوي (ت1304
، )ىػ
الفوائد البيية في تراجـ الحنفية ، عني بتصحيحو وتعميؽ الزوائد عميو السيد محمد بدر الديف أبو فراس
النعساني ، (بيروت ، دار المعرفة ، د.ت) ، ص14
؛احمد بف محمد المقري التممساني (ت1041
ىػ)، نفح
الطيب مف غصف
، األندلس الرطيب وذكر وزيرىا لساف الديف ابف الخطيب ، تحقيؽ د. االحاالت إحساف عباس
(بيروت ، دار صادر1968
، )ـ2
/
1173
؛ محمد بف عمي الشوكاني (ت1250
ىػ) ، البدر الطالع
، )بمحاسف مف بعد القرف السابع ، (بيروت ، دار المعرفة ، د.ت2
/
45
-
46
؛ صديؽ بف حسف القنوجي
(ت1307
ىػ) ، أبجد العموـ الوشي المرقوـ في بياف أحواؿ العموـ ، تحقيؽ عبد الجبار زكار ، (بيروت ، دار
، النشر العممية1978
، )ـ2
/
114
؛ جرجي زيداف ، تاريخ آداب المغة العربية ، (بيروت ، مكتبة دار
، الحياة1967
، )ـ3
/
247
؛ دائرة المعارؼ اإلسالمية ، نقميا إلى العربي ، ة احمد الشنتاوي وآخريف
، (بيروت ، دار الفكر العربي1352
/ ىػ1933
، )ـ1
/
118
،
Brockleman G. Geschicht Dear
Arabischin litteratur (Leiden, 1939: 2-274) (
2
)
عبد الرحمف بف الكماؿ جالؿ الديف السيوطي (ت911
ىػ) ، ذيؿ طبقات الحفاظ ، نشر باعتناء وستنفمد
، (غوتن ، جي1833
–
1834
، )
3
/
85
. (
4
)
، اسـ لبمدة واحدة يقاؿ ليا جزيرة عمر ، وبالد عدة منيا: الموصؿ ، سنجار ، حراف ، الرقة ، رأس عيف
آمد ، ميافارقيف ، وىي بالد بيف دجمة والفرات يقاؿ ليا الجزيرة ، أبو سعد عبد الكريـ بف محمد بف منصور
التميم ي السمعاني (ت562
ىػ) ، األنساب ، عني بتصحيحو والتعميؽ عميو عبد الرحمف بف يحيى المعممي
اليماني ، ط1
، (اليند ، وزارة المعارؼ لمتحقيقات العممية1383
/ىػ1963
،)
3
/
639
، ابف حجر: الدرر
، الكامنة1
/
256
، ، السيوطي: ذيؿ طبقات الحفاظ1
/
549
؛ مصطفى بف عبد اهلل القسطنطيني الرومي
الحنفي حاجي خميفة (ت1067
ىػ) ، كشؼ الظنوف عف أسامي الكتب والفنوف ، (بيروت ، دار الكتب
، العممية1413
/ىػ1992
، )ـ2
/
1647
؛ أبو الفالح عبد الحي بف العماد الحنبمي (ت1089
، )ىػ
.شذرات الذىب في أخبار مف ذىب ، (بيروت ، دار الكتب العممية ،د ، )ت4
/
204
؛ الشوكاني: البدر
، الطالع2
/
45
. )
996
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 ( العدد2
( ) اجمللد91
)
حزيران
2222 ( العدد2
( ) اجمللد91
)
حزيران
2222 (
5
)
، السيوطي: طبقات الحفاظ1
/
549
، ؛ ابف العماد: ـ.ف4
/
204
. (
6
)
،ابف تغري بردي: النجوـ الزاىرة14
/
276
، ؛ ألنعيمي:ـ.ف1
/
112
. االحاالت (
29
)
محمد بف عبد اهلل بف احمد بف عبد اهلل بف احمد بف محمد بف إبراىيـ بف احمد المقدسي ألصالحي
الحنبمي الشيير بابف المحب الصامت ، ولد يوـ الجمعة اوؿ رمضاف سنة712
ىػ ، خرّج وأفاد وسمع منو
الطمبة و الحفاظ ، وانتيى إليو الحفظ في زمانو ، توفي سنة789
÷
، ػ ، ابف الجزري: غاية النياية2
/
174
-
175
، ، السيوطي: ذيؿ طبقات الحفاظ1
/
366
-
367
، ؛ كحالو: معجـ المؤلفيف10
/
96
. (
29
)
محمد بف عبد اهلل بف احمد بف عبد اهلل بف احمد بف محمد بف إبراىيـ بف احمد المقدسي ألصالحي
الحنبمي الشيير بابف المحب الصامت ، ولد يوـ الجمعة اوؿ رمضاف سنة712
ىػ ، خرّج وأفاد وسمع منو
الطمبة و الحفاظ ، وانتيى إليو الحفظ في زمانو ، توفي سنة789
÷
، ػ ، ابف الجزري: غاية النياية2
/
174
-
175
، ، السيوطي: ذيؿ طبقات الحفاظ1
/
366
-
367
، ؛ كحالو: معجـ المؤلفيف10
/
96
. (
30
)
محمد بف محمد بف احمد أبو الفتح العسقالني ثـ المصري ، رحمة القراء بالديار المصرية ، ولد سنة
704
ىػ بخط جامع طولوف ، توفي سنة793
÷
:ػ بمنزلو جوار الجامع الطولوني ، ابف الجزري: ـ.ف2
/
82
، ؛ السخاوي: الضوء الالمع2
/
193
، ؛ ابف العماد: شذرات الذىب6
/
330
. (
31
)
، ابف الجزري: ـ.ف2
/
129
. ا
(
33
)
إبراىيـ ابف احمد بف عبد الواحد بف عبد المؤمف بف سعيد بف عمواف بف كامؿ أبو اسحؽ الشامي الحريري
، ولد سنة709
ىػ بدمشؽ، قرأ القراآت وغيرىا وأجازه جماعتو ، توفي سنة800
ىػ بمصر وىو آخر
، المسنديف ليا ، ابف الجزري: ـ.ف1
/
7
-
8
، ؛ السخاوي: ـ.ف2
/
193
، ، ابف العماد: ـ.ف6
/
330
. (
34
)
، ابف الجزري: ـ.ف1
/
129
. (
35
)
، السخاوي: التحفة المطيفة1
/
61
. (
36
)
:ـ.ف1
/
122
. (
36
)
:ـ.ف1
/
122
. االحاالت (
7
)
إسماعيؿ بف عمرو كثير القرشي أبو الفداء (ت774
ىػ) ، البداية والنياية في التاريخ ، (بيروت ، مكتبة
،)المعارؼ ، د.ت14
/
206
. (
8
)
، ابف حجر: الدرر الكامنة1
/
33
، ؛ السيوطي: ذيؿ طبقات الحفاظ1
/
549
، ؛ ابف العماد: ـ.ف4
/
204
. 204
. (
9
)
، ابف العماد: ـ.ف4
/
204
، ؛ دائرة المعارؼ اإلسالمية1
/
118
. (
10
)
، السيوطي: ـ.ف1
/
549
. (
9
)
، ابف العماد: ـ.ف4
/
204
، ؛ دائرة المعارؼ اإلسالمية1
/
118
. (
10
)
، السيوطي: ـ.ف1
/
549
. (
11
)
، ألنعيمي: ـ.ف1
/
245
. (
12
)
، دائرة المعارؼ اإلسالمية1
/
118
. (
12
)
، دائرة المعارؼ اإلسالمية1
/
118
. (
13
)
، ابف تغري بردي: ـ.ف14
/
276
. (
14
)
:ابف الجزري1
/
129
، ؛ السخاوي: الضوء الالمع2
/
193
؛ إسماع يؿ باشا محمد الباباني البغدادي
(ت1339ىػ) ، ىدية العارفيف-
، أسماء المؤلفيف وآثار المصنفيف ، (استانبوؿ ، وكالة المعارؼ1955
،)ـ
،
1
/
128
؛ خير الديف الزركمي: األعالـ-
قاموس تراجـ ألشير الرجاؿ والنساء مف العرب والمستعربيف
والمستشرقيف ، ط3
، (بيروت1969
، )ـ1
/
227
. (
15
)
، السيوطي: ذيؿ طبقات الحفاظ1
/
549
. (
16
)
،ابف العماد: ـ.ف4
/
204
، ؛ يوسؼ آلياف سركيس: معجـ المطبوعات العربية والمعربة ، (القاىرة
، سركيس1346
ىػ) ، ص ص62
-
63
. (
24
)
، شذرات الذىب4
/
205
. (
25
)
، ابف الجزري: غاية النياية1
/
129
-
130
. ـ
(
27
)
:ـ.ف1
/
159
، ؛ السخاوي: الضوء الالمع2
/
193
؛ واإل جازة عبارة عف آذاف الشيخ لتمميذه برؤية
مسموعاتو ومؤلفاتو ساء التي سمعيا مف شيخو أو التي لـ يسمعيا منو ولـ يقرأىا عميو ، السخاوي: فتح )
997
) ( العدد2
( ) اجمللد91
)
حزيران
2222 ، المغيث بشرح ألفية الحديث ، تحقيؽ عمي حسني عمي ؛ (اليند، الجامعة السمفية1407
)
3
/
88
؛
، صبحي الصالح: عموـ الحديث ومصطمحو
، (دمشؽ ، جامعة دمشؽ1379
/ىػ1959
ـ) ، ص94
. (
28
)
:ـ.ف1
/
129
. االحاالت ـ
(
41
)
:ـ.ف2
/
403
-
404
–
واإلمالء مف أساليب التعميـ في التربية اإلسالمية، وكانت تعقد لو المجالس
ويممي فييا الشيخ مف حفظو ، لذلؾ عدّ اإلمالء أعمى مراتب السماع وفيو أحسف وجوه التحمؿ وأقواىا ال
يتصدى لو إال المحدث العارؼ ، السيوطي: تدريب الراوي في شرح تقريب النواوي ، تحقيؽ عبد الوىاب عبد
، المطيؼ ، (بيروت ، دار إحياء الكتب1979
، )ـ2
/
132
فما بعدىا ؛ شاكر محمود عبد المنعـ
، العسقالني دراسة مصنفاتو ومنيجو وموارده في كتابو اإلصابة ، (جامعة بغداد ، كمية اآلداب1976ـ-
)رسالة دكتوراه1
/
212
. (
42
)
، ألنعيمي: الدارس1
/
460
. (
43
)
، شذرات الذىب4
/
165
. (
43
)
، شذرات الذىب4
/
165
. )
998
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 (
44
)
:ـ.ف4
/
186
. (
45
)
:الدارس4
/
223
. (
46
)
ابو عمر وعثماف بف الصالح والشيرزوري (ت643
، ىػ) ، عموـ الحديث ، تحقيؽ نور الديف عتر
(بيروت ، المكتبة العممية ، د.ت) ، ص222
؛ ابو بكر احمد بف عمي الخطيب البغدادي (ت463
، )ىػ
الرحمة في
طمب الحديث ، تحقيؽ نور الديف عتر ، (بيروت، دار الكتب العممية ، د. ت) ، ص16
وما
.بعدىا (
47
)
، ابف الجزري: غاية النياية1
/
129
. (
48
)
.ـ.ف
(
49
)
:ـ.ف1
/
130
–
حصمت الوقعة التيمورية في سنة799
ىػ ، إذ أخذ عسكر تيمورلنؾ ارزنجاف وقتؿ
أىميا ونيب ما فييا ، فمما بم غ سمطاف مصر والشاـ الظاىر برقوؽ ذلؾ أرسؿ نوابو في الشاـ أف يتوجيوا إلى
شاطئ الفرات فخرجوا كميـ وأقاموا ىناؾ فمما اشرفوا عمى سيواس انيزـ التتار منيـ فقصده قرايموؾ بف عمي
، التركي أواخر سنة ثمانمائة فانكسر عسكر سيواس ، السخاوي: الضوء الالمع1
/
371
؛ محمد كرد :
خطط الشاـ ، ط2
( ، (بيروت ، دار العمـ لممالييف1391
/ ىػ1971
، )ـ2
/
163
. (
50
)
-
:ـ.ف1
/
130
. االحاالت (
51
)
، ابف الجزري: غاية النياية1
/
130
–
الممؾ االشرؼ برسباي ىو سيؼ الديف ابو النصر برسباي
الدقماقي ، بويع بالسمطنة سنة825
ىػ ، فكانت مدة واليتو ست عشرة سنة وثمانية أشير ، بدر الديف محمود
بف احمد العيني (ت855
ىػ) ، الروض الزاىر في سير الممؾ الظاىر ططر ، تحقي ، ؽ ىانس ارنست
، (القاىرة ، دار إحياء الكتب العربية1962ـ) ، ص2
؛ عبد الممؾ بف حسيف بف عبد الممؾ العصامي
المكي (ت1049
، )ىػ) ، سمط النجوـ العوالي في أنباء األوائؿ والتوالي ، (القاىرة ، السمفية ، د.ت4
/
38
-
39
. (
49
)
:ـ.ف1
/
130
–
حصمت الوقعة التيمورية في سنة799
ىػ ، إذ أخذ عسكر تيمورلنؾ ارزنجاف وقتؿ
أىميا ونيب ما فييا ، فمما بم غ سمطاف مصر والشاـ الظاىر برقوؽ ذلؾ أرسؿ نوابو في الشاـ أف يتوجيوا إلى
شاطئ الفرات فخرجوا كميـ وأقاموا ىناؾ فمما اشرفوا عمى سيواس انيزـ التتار منيـ فقصده قرايموؾ بف عمي
، التركي أواخر سنة ثمانمائة فانكسر عسكر سيواس ، السخاوي: الضوء الالمع1
/
371
؛ محمد كرد :
خطط الشاـ ، ط2
( ، (بيروت ، دار العمـ لممالييف1391
/ ىػ1971
، )ـ2
/
163
. (
) (
51
)
، ابف الجزري: غاية النياية1
/
130
–
الممؾ االشرؼ برسباي ىو سيؼ الديف ابو النصر برسباي
الدقماقي ، بويع بالسمطنة سنة825
ىػ ، فكانت مدة واليتو ست عشرة سنة وثمانية أشير ، بدر الديف محمود
بف احمد العيني (ت855
ىػ) ، الروض الزاىر في سير الممؾ الظاىر ططر ، تحقي ، ؽ ىانس ارنست
، (القاىرة ، دار إحياء الكتب العربية1962ـ) ، ص2
؛ عبد الممؾ بف حسيف بف عبد الممؾ العصامي
المكي (ت1049
، )ىػ) ، سمط النجوـ العوالي في أنباء األوائؿ والتوالي ، (القاىرة ، السمفية ، د.ت4
/
38
-
39
. (
53
) ابف العماد ، : شذرات الذىب4
/
204
. )
999
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 ( العدد2
( ) اجمللد91
)
حزيران
2222 (
60
)
مطبوع–
مف الكتب الجامعة لألدعية واألوراد واألذكار الواردة في األحاديث واآلثار ، فرغ مف تأليفو
سنة791
ىػ في دمشؽ ، طبع حجر في مصر1277
ص160
، بوالؽ1320
، وبيامش كتاب خزينة
األسرار جميمة األذكار لمحمد حقي النازلي ، سركيس معجـ المطبوعات ، ص63
. (
61
) مخطوط-
، الزركمي: ـ.ف7
/
275
. (118
-
،ـ.ف1
/
70
. (123
-
،ـ.ف2
/
1444
. االحاالت )
1001
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 )
1002
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 (139
-
،ـ.ف1
/
87
. (140
-
،ـ.ف1
/
388
. (141
-
،ـ.ف2
/
44
. (142
-
،ـ.ف1
/
270
. (143
-
،ـ.ف1
/
274
. (144
-
،ـ.ف1
/
324
. (145
-
،ـ.ف1
/
272
. (146
-
،ـ.ف1
/
48
. (147
-
،ـ.ف1
/
185
. (148
-
،ـ.ف1
/
164
. (149
-
،ـ.ف1
/
247
. (150
-
،ـ.ف1
/
183
. (151
-
،ـ.ف1
/
187
. (152
-
،ـ.ف1
/
194
. (153
-
،ـ.ف1
/
69
؛
عيسى بؾ :احمد،تاريخ البيمارستانات في اإلسالـ،ط2
،( بيروت،دار الرائد العربي
1401
/ىػ1981
)ـ-
))((يوضح في ىذا الكتاب المصطمحات العممية لالختصاصات الطبية آنذاؾ
(154
-
،ـ.ف1
/
343
. (155
-
،ـ.ف1
/
344
. (156
-
،ـ.ف2
/
33
. (157
-
،ـ.ف1
/
344
. (158
-
،ابف الجزري:ـ.ف1
/
353
-
354
. (159
-
،ـ.ف1
/
388
. (160
-
،ـ.ف2
/
49
. (161
-
،مـ.ف1
/
83
. (162
-
،ـ.ف1
/
388
. (ـ ف/
(153
-
،ـ.ف1
/
69
؛
عيسى بؾ :احمد،تاريخ البيمارستانات في اإلسالـ،ط2
،( بيروت،دار الرائد العربي
1401
/ىػ1981
)ـ-
))((يوضح في ىذا الكتاب المصطمحات العممية لالختصاصات الطبية آنذاؾ
(154
-
،ـ.ف1
/
343
. (ـ
(153
-
،ـ.ف1
/
69
؛
عيسى بؾ :احمد،تاريخ البيمارستانات في اإلسالـ،ط2
،( بيروت،دار الرائد العربي
1401
/ىػ1981
)ـ-
))((يوضح في ىذا الكتاب المصطمحات العممية لالختصاصات الطبية آنذاؾ
(154
-
،ـ.ف1
/
343
. (155
-
،ـ.ف1
/
344
. (158
-
،ابف الجزري:ـ.ف1
/
353
-
354
. االحاالت اإ
(
63
)
لتتميـ القراآت العشر ، نظميا تكممة لمشاطبية عمى وزنيا ورد بيا في مجموعة رقـ86
، ، (القاىرة
1308
/ ىػ1890
ـ) ، سركيس: ـ.ف، ص63
، ؛ عبد الجبار: ذخائر التراث1
/
71
. (
64
)
مطبوع–
ا و المقدمة فيما يجب عمى القارئ أف يعممو وتعرؼ بالجزرية منظومة مف7
أبيات في
التجويد ، سركيس: ـ.ف ، ص63
. (
65
)
، الزركمي: ـ.ف7
/
275
. (
65
)
، الزركمي: ـ.ف7
/
275
. (
67
)
مطبوع ثالث طبعات–
مط عبد الرزاؽ1305
ىػ، ص64
، مط الميمنة1310
ىػ، ص63
؛ العممية
1313
ى ،ػ؛ عبد الجبار: ذخائر التراث1
/
71
. (
68
)
، مطبوع (بوالؽ1309
/ ىػ1891
ـ) ، ص64
،؛ عبد الجبار: ـ.ف1
/
7
. (
69
)
، شرح حسنيف مخموؼ (القاىرة ، لجنة البياف العربي1961
ـ) ،ص20
.، ـ.ف
(
70
) مطبوع-
،(القاىرة، السعادة1347
/ ىػ1938
.ـ)، جزئييف، ـ.ف (
72
)
مطبوع–
، تحقيؽ عبد الحي الغرقاوي (القاىرة، مكتبة القدس1305
/ىػ1931
ـ) ، ص79
؛
، (القاىرة ، مكتبة جميورية مصر1977
ـ) ، ص296
..ـ.ف
(73
-
،ابف الجزري:ـ.ف1
/
94
. (77
-
،ـ.ف1
/
511
. (78
-
،ـ.ف2
/
211
. )
1000
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 -
،ـ.ف1
/
91
،
168
،
169
،
243
. (93
-
،ـ.ف1
/
324
. (94
-
،ـ.ف1
/
169
. (95
-
،ـ.ف1
/
551
. (96
-
،ـ.ف1
/
69
. (97
-
،ـ.ف1
/
457
. (101
-
،ـ.ف1
/
45
. (102
-
:ابف الجزري،ـ.ف1
/
70
. (103
-
،ـ.ف1
/
79
. (ـ
(104
-
نسبة إلى ألشروطي،أي نسبة إلى كتب الوثائؽ بالديواف والبيانات،أما الشروط في الوثائؽ التي تكتب
في السجالت الخاصة في حضرة القاضي وتثبيت فييا األحكاـ التي يصدرىا القاضي، القمقشندي: تقي الديف
احمد بف عمي (ت821ىػ)، صبح األعشى في صناعة االنشا،( القاىرة،المؤسسة
المصرية العامة لمتأليؼ
،)والترجمة والطباعة والنشر5
/
466؛معروؼ:ناجي،عمماء النظاميات ومدارس المشرؽ اإلسالمي ،ط1
(
،بغداد،اإلرشاد1393
/ىػ1973ـ)،ص245
. إ
(105
-
:ومفردىا الشاىد أو العدؿ أو المعدؿ،وىو الذي يشيد بمتعمقات الديواف نفيا وا ثباتا ،القمقشندي
،ـ.ف5
/
466
. (106
-
،ابف الجزري:ـ.ف1
/
45
. (107
-
،ـ.ف1
/
45
. (108
-
،ـ.ف1
/
241
. English Reference English Reference Al-Baghdadi: Ismail Muhammad al-Babani (d. 1339 Ah) 1-the gift of the knowledgeable / the names of the authors and the effects of
the classifiers, (Istanbul, knowledge agency, 1955). Ibn taghri Bardi: Abu al-Muhassin Yusuf ibn taghri Bardi Al-Atabaki (d. 874 Ah) Ibn taghri Bardi: Abu al-Muhassin Yusuf ibn taghri Bardi Al-Atabaki (d. 874 Ah) )
1003
) )
1003
) ( العدد2
( ) اجمللد91
)
حزيران
2222 ( العدد2
( ) اجمللد91
)
حزيران
2222 جملة جامعة االنبار للعلوم االنسانية 2-the blooming stars of the Kings of Egypt and Cairo, (Cairo, Egyptian
General Organization for authorship, translation, printing and Publishing,
Dr.C). 2-the blooming stars of the Kings of Egypt and Cairo, (Cairo, Egyptian
General Organization for authorship, translation, printing and Publishing,
Dr.C). 2-the blooming stars of the Kings of Egypt and Cairo, (Cairo, Egyptian
General Organization for authorship, translation, printing and Publishing,
Dr.C). Ibn al-Jazari: Ahmad ibn Muhammad ibn Ali ibn Yusuf ibn al-Jazari (d. 833 Ah) Ibn al-Jazari: Ahmad ibn Muhammad ibn Ali ibn Yusuf ibn al-Jazari (d. 833 Ah) 3-the end of the layers of readers, published by pragstrasser, (Cairo, Al-
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( ) اجمللد91
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ATAR, (Beirut, scientific books House, Dr.C). 10-flags-a dictionary of translations of the most famous men and women
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R Ibn Kathir: Ismail ibn Amr Ibn Kathir al-Qurashi Abu al-Fida (d. 774 Ah). -the beginning and the end in history, (Beirut, knowledge library, D.C). A
O
R -Dictionary of authors / translations of Arabic book compilers, (Beirut,
1957). -Dictionary of authors / translations of Arabic book compilers, (Beirut,
1957). -plans of al-Sham, i2 (Beirut, Dar Al-Alam for millions, 1391 Ah/ 1971 ad). Known:Bashar Awad. -plans of al-Sham, i2 (Beirut, Dar Al-Alam for millions, 1391 Ah/ 1971 ad). Known:Bashar Awad. 28-al-dhahabi and his methodology in writing the history of Islam, Vol.1(
Isa Al-Babi al-Halabi, 1976). 28-al-dhahabi and his methodology in writing the history of Islam, Vol.1(
Isa Al-Babi al-Halabi, 1976). Al-maqri Al-tlemsani: Ahmad ibn Muhammad (d. 1041 Ah). )
1005
) جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222
Blowing the Tayeb from the damp branch of Andalusia and reminded her
minister of San al-Din al-Khatib, the investigation of Dr. Ihsan Abbas,
(Beirut, Dar Sadr , 1968). Brockleman G. Geschichte Der Arabischin Lettertur (Leiden, 1939). جملة جامعة االنبار للعلوم االنسانية
( العدد2
( ) اجمللد91
)
حزيران
2222 ( العدد2
( ) اجمللد91
)
حزيران
2222 Blowing the Tayeb from the damp branch of Andalusia and reminded her
minister of San al-Din al-Khatib, the investigation of Dr. Ihsan Abbas,
(Beirut, Dar Sadr , 1968). 1- )
1006
)
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Structural changes in water and Ar-water clusters under high pressure
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My IOPscience Structural changes in water and Ar-water clusters under high pressure This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 J. Phys.: Conf. Ser. 635 032008
(http://iopscience.iop.org/1742-6596/635/3/032008) View the table of contents for this issue, or go to the journal homepage for more Download details: R. Prosmiti† 1, A. V´ıtek∗, D.J. Arismendi-Arrieta†, R. Rodriguez-Cantano†,
P. Villarreal†, R. Kalus∗, G. Delgado-Barrio† For the pure water (H2O)20 clus-
ter, at low pressures such transitions correspond
from the “all-surface” structures to the cage-1
like structures, while at higher pressures transi-
tions to the cage-1 isomeric structure. For the
Ar(H2O)20 cluster, changes at high pressure cor-
respond to solid-to-solid transitions from outside-
to-inside structures, and have been related to
clathrate-like cages around the Ar atom. It is
also shown that the formation and thermody-
namic stability of the regular 512 structures are
determined by the intermolecular interaction be- 200
400
600
800
C
B
10 GPa
1GPa
100 MPa
10 MPa
1MPa
100 kPa
P
T (K)
10 kPa
A (
)
Figure 1. Upper panel: Potentials as a function of
the distance between the Ar atom and the center of
mass of the 512 cage formed by 20 H2O molecules
(see inset plot). Lower panel: Phase diagrams of
the Ar(H2O)20 cluster. Black dot lines correspond
to the maxima of the heat capacity, blue/red dot
lines to the min/max of the Pearson correlation co-
efficient, and A, B, and C to the different solid–solid
transitions. Figure 1. Upper panel: Potentials as a function of
the distance between the Ar atom and the center of
mass of the 512 cage formed by 20 H2O molecules
(see inset plot). Lower panel: Phase diagrams of
the Ar(H2O)20 cluster. Black dot lines correspond
to the maxima of the heat capacity, blue/red dot
lines to the min/max of the Pearson correlation co-
efficient, and A, B, and C to the different solid–solid
transitions. Figure 1. Upper panel: Potentials as a function of Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd
1 XXIX International Conference on Photonic, Electronic, and Atomic Collisions (ICPEAC2015)
IOP Publishing
Journal of Physics: Conference Series 635 (2015) 032008
doi:10.1088/1742-6596/635/3/032008 X International Conference on Photonic, Electronic, and Atomic Collisions (ICPEAC2015)
IOP Publishin
al of Physics: Conference Series 635 (2015) 032008
doi:10.1088/1742-6596/635/3/03200 IP Address: 161.111.22.36
This content was downloaded on 22/09/2015 at 10:51 Please note that terms and conditions apply. R. Prosmiti† 1, A. V´ıtek∗, D.J. Arismendi-Arrieta†, R. Rodriguez-Cantano†,
P. Villarreal†, R. Kalus∗, G. Delgado-Barrio† † Institute of Fundamental Physics, IFF-CSIC, Serrano 123, 28006 Madrid, Spain
∗IT4Innovations National Supercomputing Center, and Department of Applied Mathematics, VSB–Technical
University of Ostrava, 70833 Ostrava, Czech Republic Synopsis Specific size gas-water clusters are currently receiving considerable attention, as models for inclusion
compounds of different type of clathrate hydrates. As model microsolutions they retain many characteristics of
the bulk, are theoretically tractable, and can be used to probe the relevant guest/host interactions, as well as to
derive and to test intermolecular potentials that can be also used under different thermodynamic conditions. tween the Ar atom and host water network [2]. A number of spectroscopic and diffraction
studies have been conducted to explore at which
conditions bulk rare-gas hydrates may be formed
or decomposed and what types of them may be
obtained at such conditions [1]. Theoretical at-
tempts to describe structural transitions in these
clathrate hydrates could provide useful informa-
tion, and could serve to settle disagreement be-
tween different sets of experiments. x
y
z
200
400
600
800
C
B
10 GPa
1GPa
100 MPa
10 MPa
1MPa
100 kPa
P
T (K)
10 kPa
A
Figure 1. Upper panel: Potentials as a function of
the distance between the Ar atom and the center of
mass of the 512 cage formed by 20 H2O molecules
(see inset plot). Lower panel: Phase diagrams of
the Ar(H2O)20 cluster. Black dot lines correspond
to the maxima of the heat capacity, blue/red dot
lines to the min/max of the Pearson correlation co-
efficient, and A, B, and C to the different solid–solid
transitions. x
y
z Thus, we performed [2] parallel tempering Monte
Carlo
simulations
in
the
isothermal-isobaric
(NPT) ensemble, aiming to provide a complete
description of the cluster phase behavior under
various temperature–pressure conditions. The
TIP4P/ice model was employed for the water-
water interactions, while both semiempirical and
ab initio-based potentials were used to model the
interaction between the rare-gas atom and water
molecules (see Fig. 1). We constructed heat ca-
pacity phase diagrams for specific size clusters,
such as the small polyhedral cages of the sI, sII
and sH clathrate structures. By analyzing the
heat capacity landscape and the Pearson corre-
lation coefficient profile for the interaction energy
and volume, structural changes were detected [2]
(see Fig. 1). [1] J.S. Loveday et al 2008 Phys. Chem. Chem. Phys.
10 937 1E-mail: rita@iff.csic.es References [1] J.S. Loveday et al 2008 Phys. Chem. Chem. Phys. 10 937 [1] J.S. Loveday et al 2008 Phys. Chem. Chem. Phys. 10 937 [2] A. Vitek et al 2015 Phys. Chem. Chem. Phys. DOI: 10.1039/c4cp04862h [2] A. Vitek et al 2015 Phys. Chem. Chem. Phys. DOI: 10.1039/c4cp04862h 1E-mail: rita@iff.csic.es 1E-mail: rita@iff.csic.es
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https://openalex.org/W3156667917
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https://peerj.com/articles/11053v0.3/submission
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English
| null |
Peer Review #2 of "Effect of cotton residues incorporation on soil properties, organic nitrogen fractions, and nitrogen-mineralizing enzyme activity under long-term continuous cotton cropping (v0.2)"
| null | 2,021
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Manuscript to be reviewed Manuscript to be reviewed 1
Effect of cotton residues incorporation on soil properties, organic nitrogen fractions, and
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nitrogen-mineralizing enzyme activity under long-term continuous cotton cropping
3
Fangxia Maa, Yiyun Wang, Peng Yan, Fei Wei, Zhiping Duan, Zhilan Yang & Jianguo Liu*
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a The Key Laboratory of Oasis Ecology Agriculture of Xinjiang Bingtuan, Shihezi University,
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Shihezi, Xinjiang, China. 7
8
*Corresponding author. E-mail: l-jianguo@126.com
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Running Head: cotton residues dose organic nitrogen and enzyme activity
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Effect of cotton residues incorporation on soil properties, organic nitrogen f
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nitrogen-mineralizing enzyme activity under long-term continuous cotto
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Fangxia Maa, Yiyun Wang, Peng Yan, Fei Wei, Zhiping Duan, Zhilan Yang &
4
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a The Key Laboratory of Oasis Ecology Agriculture of Xinjiang Bingtuan, Shih
6
Shihezi, Xinjiang, China. 7
8
*Corresponding author. E-mail: l-jianguo@126.com
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Running Head: cotton residues dose organic nitrogen and enzyme activity
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Abstract Effect of cotton residues incorporation on soil properties,
organic nitrogen fractions, and nitrogen-mineralizing enzyme
activity under long-term continuous cotton cropping
Fangxia Ma
1 , Yiyun Wang
1 , Peng Yan
1 , Fei Wei
1 , Zhiping Duan
1 , Zhilan Yang
1 , Jianguo Liu
Corresp.
1
1 The Key Laboratory of Oasis Ecology Agriculture of Xinjiang Bingtuan, Shihezi University, Shihezi, Xinjiang, China The objective of this experiment was to study the effect of cotton residues incorporation
on soil properties, soil organic nitrogen (N) fractions, and N-mineralizing enzyme (protease,
and urease) activity in the 0-40 cm soil layer in the long-term continuous cotton field. In
this experiment, seven treatments, including cotton residues incorporation for 5, 10, 15
and 20 years (marked as 5a, 10a, 15a, and 20a) and continuous cropping for 5, 10 and 20
years (marked as CK5, CK10 and CK20) were conducted. The results showed that the soil
organic carbon (C) and N increased gradually with the increase in the duration of
continuous cropping with cotton residues incorporation. Compared with CK20, the 20a
treatments reduced the content of amino acid N (AAN), ammonium N (AN), amino sugar N
(ASN), hydrolysable unidentified N (HUN), and acid insoluble N (AIN) significantly by 48.6,
32.2, 96.9, 48.3, and 38.7%, respectively (p<0.05). The activity of protease and urease in
20a treatments significantly increased by 53.4 and 53.1% respectively as compared to
CK20 (p<0.05). Soil organic C and N-mineralizing enzyme activity decreased with the
increase in cropping duration in the absence of cotton residues incorporation, while the
organic N increased slightly. In conclusion, cotton residues returning can increase the
storage of soil organic C and N in long-term continuous cropping cotton field, and improve
the soil quality and soil fertility of continuous cropping cotton field. PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed 1
Effect of cotton residues incorporation on soil properties, organic nitrogen fractions, and
2
nitrogen-mineralizing enzyme activity under long-term continuous cotton cropping 5
a The Key Laboratory of Oasis Ecology Agriculture of Xinjiang Bingtuan, Shihezi University,
6
Shihezi, Xinjiang, China. 5
a The Key Laboratory of Oasis Ecology Agriculture of Xinjiang Bingtuan, Shihezi University,
6
Shihezi, Xinjiang, China. 21
Abstract PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed 22
The objective of this experiment was to study the effect of cotton residues incorporation on soil
23
properties, soil organic nitrogen (N) fractions, and N-mineralizing enzyme (protease, and urease)
24
activity in the 0-40 cm soil layer in the long-term continuous cotton field. In this experiment,
25
seven treatments, including cotton residues incorporation for 5, 10, 15 and 20 years (marked as
26
5a, 10a, 15a, and 20a) and continuous cropping for 5, 10 and 20 years (marked as CK5, CK10
27
and CK20) were conducted. The results showed that the soil organic carbon (C) and N increased
28
gradually with the increase in the duration of continuous cropping with cotton residues
29
incorporation. Compared with CK20, the 20a treatments reduced the content of amino acid N
30
(AAN), ammonium N (AN), amino sugar N (ASN), hydrolysable unidentified N (HUN), and
31
acid insoluble N (AIN) significantly by 48.6, 32.2, 96.9, 48.3, and 38.7%, respectively (p<0.05). 32
The activity of protease and urease in 20a treatments significantly increased by 53.4 and 53.1%
33
respectively as compared to CK20 (p<0.05). Soil organic C and N-mineralizing enzyme activity
34
decreased with the increase in cropping duration in the absence of cotton residues incorporation,
35
while the organic N increased slightly. In conclusion, cotton residues returning can increase the
36
storage of soil organic C and N in long-term continuous cropping cotton field, and improve the
37
soil quality and soil fertility of continuous cropping cotton field. 38
Keywords：Cotton residues incorporation; organic N fractions; N-mineralizing enzymes;
39
continuous cotton cropping
40
Introduction
41
Nitrogen (N) is an essential nutrient for crop growth and net primary productivity (Mulvaney, 38
Keywords：Cotton residues incorporation; organic N fractions; N-mineralizing enzymes;
39
continuous cotton cropping 38
Keywords：Cotton residues incorporation; organic N fractions; N-mineralizing enzymes;
39
continuous cotton cropping 41
Nitrogen (N) is an essential nutrient for crop growth and net primary productivity (Mulvaney,
42
2009). As over 90% soil N is existed in organic forms, soil N availability was primarily PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed 43
determined by soil organic carbon (C) and N (Stevenson, 1982). Crops primarily take up
44
inorganic N and can take in a small part of low molecular weight organic N under extreme
45
conditions (Ashton et al., 2010). Therefore, the mineralization of organic N is a critical
46
parameter regulating ecosystem productivity (Keuper et al., 2017). 43
determined by soil organic carbon (C) and N (Stevenson, 1982). Crops primarily take up
44
inorganic N and can take in a small part of low molecular weight organic N under extreme
45
conditions (Ashton et al., 2010). Therefore, the mineralization of organic N is a critical
46
parameter regulating ecosystem productivity (Keuper et al., 2017). 47
The mineralization and depolymerization of organic N in the soil involves a sequence of
48
microbial enzymatic processes (Mengel, 1996). Most of the N input into the soil is in the form of
49
polymers, which must first be decomposed into smaller units by extracellular enzymes (Schimel
50
et al., 2004), which release small organic molecules that can then be directly absorbed or go on
51
degraded, N is absorbed in the form of ammonium (NH4+). Proteases are the most important
52
extracellular depolymerases for the hydrolysis of N-containing molecules, and large proteins and
53
peptides can be hydrolyzed into peptides and amino acids, its activity is closely related to
54
microbial activity (Geisseler et al., 2008). Urease, which released ammonia from linear amides is
55
also an important depolymerase. 47
The mineralization and depolymerization of organic N in the soil involves a sequence of
48
microbial enzymatic processes (Mengel, 1996). Most of the N input into the soil is in the form of
49
polymers, which must first be decomposed into smaller units by extracellular enzymes (Schimel
50
et al., 2004), which release small organic molecules that can then be directly absorbed or go on
51
degraded, N is absorbed in the form of ammonium (NH4+). Proteases are the most important
52
extracellular depolymerases for the hydrolysis of N-containing molecules, and large proteins and
53
peptides can be hydrolyzed into peptides and amino acids, its activity is closely related to
54
microbial activity (Geisseler et al., 2008). Urease, which released ammonia from linear amides is
55
also an important depolymerase. Manuscript to be reviewed 64
primary means of organic fertilization in this region. Malhi et al. (2011) found that cotton
65
residues incorporation could increase the content of organic N under conventional and
66
conservational tillage conditions and could also reduce N losses through microbial N
67
sequestration (Shan et al., 2013). However, little research has been done to elucidate the response
68
of soil organic N pools and N-mineralizing enzyme activity for long-term continuous cropping
69
and cotton residues incorporation. The objective of this experiment was to study the effects of
70
cotton residues incorporation on soil properties, soil organic N fractions and the relationship
71
between soil organic N fractions and N-mineralizing enzyme activity in a long-term continuous
72
cotton cropping system. We postulated that cotton residues incorporation contributes to the
73
increase in the storage of soil organic C and N in long-term continuous cropping cotton field. Manuscript to be reviewed 56
As an important source of soil organic matter (SOM), crop residues are rich in C, N, P, K
57
and trace elements (Malhi et al., 2011). The incorporation of crop residues into the soil is a
58
important option for improving soil fertility. Bakht et al. (2009) found that crop residu
59
management increased soil N, it helps maintain farmland fertility, and reduce fertilizer utilizatio
60
Govaerts et al. (2007) also found that residue management improved soil microbial biomass an
61
catabolic diversity. 62
Xinjiang is the primary cotton cultivation region in China. The perennial, continuou
63
cropping of cotton and full incorporation of cotton residues into the field have become th
PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) 56
As an important source of soil organic matter (SOM), crop residues are rich in C, N, P, K,
57
and trace elements (Malhi et al., 2011). The incorporation of crop residues into the soil is an
58
important option for improving soil fertility. Bakht et al. (2009) found that crop residue
59
management increased soil N, it helps maintain farmland fertility, and reduce fertilizer utilization. 60
Govaerts et al. (2007) also found that residue management improved soil microbial biomass and
61
catabolic diversity. 62
Xinjiang is the primary cotton cultivation region in China. The perennial, continuous
63
cropping of cotton and full incorporation of cotton residues into the field have become the 62
Xinjiang is the primary cotton cultivation region in China. The perennial, continuous
63
cropping of cotton and full incorporation of cotton residues into the field have become the 62
Xinjiang is the primary cotton cultivation region in China. The perennial, continuous
63
cropping of cotton and full incorporation of cotton residues into the field have become the PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) 75
Study sites and experimental design 76
This experiment was conducted at a long-term continuous cropping experimental field at the
77
Shihezi University Agriculture College experimental station (86°03'E, 45°19'N) situated in
78
Shihezi City, Xinjiang Uyghur Autonomous Region, China. The soil texture is gray desert soil,
79
and the basic soil properties are shown in Table 1. 80
There were seven treatments in the continuous cropping plots; four of which were cotton
81
residues incorporation treatments, the others were cotton residues removed treatments. There
82
were three replicates per treatment. The experimental design is single factor completely random
83
design. The duration of continuous cropping with cotton residues incorporation treatments
84
included 5, 10, 15, and 20 years, which were marked as 5a, 10a, 15a, and 20a, respectively. The Manuscript to be reviewed 85
continuous cropping without residues incorporation treatments included 5, 10, and 20 years,
86
which were marked as CK5, CK10, and CK20, respectively. The mode of cotton residues
87
returning to the field for continuous cropping by cutting all the cotton residues into 5-8 cm with a
88
guillotine after harvesting every autumn, namely, simulate the way of returning the cotton
89
residues to the field mechanically, turn it into the plough layer before winter, and then irrigate it
90
in the winter. The pattern of continuous cropping without cotton residues incorporation is to take
91
all the cotton residues out of the field after harvesting, and then apply chemical fertilizer,
92
ploughing, winter irrigation. The cotton was hand harvested. The average contents of C, N, P and
93
K in cotton residues were about 41.3%, 1.69%, 0.43% and 3.14% (by dry weight), respectively. 94
Nitrogen 300 kg hm-2, phosphorus (P2O5) 150 kg hm-2, potassium (K2O) 75 kg hm-2, 30% of N
95
fertilizer and 100% of phosphorus and potassium fertilizer were applied with tillage after cotton
96
harvest. The other 70% N fertilizer was applied with water irrigating. The cultivar of cotton,
97
Xinluzao 46, was sowed in April. On July, spraying growth regulator and manual topping are
98
used to control excessive growth, prevent lodging, increase earliness, and decrease cotton
99
bollworm infestations in cotton field. The way of planting was according to “30+60+30”
100
configuration, using drip irrigation under mulch. The date of sowing was 20th April, and spraying
101
growth regulator and manual topping were on 10th July every year. During the growth period, we
102
would drip 11 times, the total drip was 5400 m3 hm-2. Other field management followed the local
103
practice. 104
Soil sampling
105
Soil samples in the 0-40 cm soil layer were collected in April 2017 five sampling points were 85
continuous cropping without residues incorporation treatments included 5, 10, and 20 years,
86
which were marked as CK5, CK10, and CK20, respectively. The mode of cotton residues
87
returning to the field for continuous cropping by cutting all the cotton residues into 5-8 cm with a
88
guillotine after harvesting every autumn, namely, simulate the way of returning the cotton
89
residues to the field mechanically, turn it into the plough layer before winter, and then irrigate it
90
in the winter. Manuscript to be reviewed The pattern of continuous cropping without cotton residues incorporation is to take
91
all the cotton residues out of the field after harvesting, and then apply chemical fertilizer,
92
ploughing, winter irrigation. The cotton was hand harvested. The average contents of C, N, P and
93
K in cotton residues were about 41.3%, 1.69%, 0.43% and 3.14% (by dry weight), respectively. 94
Nitrogen 300 kg hm-2, phosphorus (P2O5) 150 kg hm-2, potassium (K2O) 75 kg hm-2, 30% of N
95
fertilizer and 100% of phosphorus and potassium fertilizer were applied with tillage after cotton
96
harvest. The other 70% N fertilizer was applied with water irrigating. The cultivar of cotton,
97
Xinluzao 46, was sowed in April. On July, spraying growth regulator and manual topping are
98
used to control excessive growth, prevent lodging, increase earliness, and decrease cotton
99
bollworm infestations in cotton field. The way of planting was according to “30+60+30”
100
configuration, using drip irrigation under mulch. The date of sowing was 20th April, and spraying
101
growth regulator and manual topping were on 10th July every year. During the growth period, we
102
would drip 11 times, the total drip was 5400 m3 hm-2. Other field management followed the local
103
practice. 104
Soil sampling 105
Soil samples in the 0-40 cm soil layer were collected in April 2017, five sampling points were 105
Soil samples in the 0-40 cm soil layer were collected in April 2017, five sampling points were Manuscript to be reviewed 106
combined to form one sample following the "S" shape in each experimental plot. After removing
107
the visible plant roots and stones, the collected soil samples were mixed and passed through a 2
108
mm sieve. One portion of the samples were stored at 4°C in order to measure the soil microbial
109
biomass C (Cmic), N (Nmic), and enzyme activity. The other soil samples were air dried and
110
ground through a 0.149 mm sieve so as to determine the content of soil organic C, total C, total
111
N, and organic N fractions. 113
Soil organic carbon (SOC) content was determined according to the method of Nelson et al. 114
(1982). The content of total organic carbon (Ctot) and total nitrogen (Ntot) in soil was measured
115
by K2Cr2O7 oxidation method and Kjeldahl method. The Soil Cmic content and soil Nmic content
116
were measured by chloroform fumigation extraction method (Joergensen, 1996). 117
Soil organic N fractions and N-mineralizing enzyme activities 117
Soil organic N fractions and N-mineralizing enzyme activities 117
Soil organic N fractions and N-mineralizing enzyme activities 118
The fractions of organic N in soils were determined by hydrolyzing soil samples with 6 M HCl
119
on an electric hot plate at 120℃ ± 3℃ for 12 h (Bremner, 1965). The content of total
120
hydrolysable N, amino acid N, and hydrolysable ammonium N was measured by the method of
121
steam distillation. The equation (1) and (2) was used to calculate the content of hydrolysable
122
unidentified N and acid insoluble N, respectively. The activities of soil protease, and urease
123
activities were measured by using colorimetrical methods. For the measurement of soil protease
124
activity, 4 g of soil samples, 20 mL 20% casein solution, and 1 mL methylbenzene was added
125
into 50ml glass triangular bottle and incubated for 24 hours at 30℃. Then the solution was
126
mixed with Sulfuric acid and sodium sulfate solutions to precipitate protein. After centrifuging, Manuscript to be reviewed 127
2ml of the supernatant and ninhydrin solution mixed in the water bath kettle at 100℃ for 1
128
minutes and finally determined at 500 nm. For the measurement of urease activity, 5g of so
129
samples and 1ml methylbenzene were added into 100 mL volumetric flask and incubated for 1
130
minutes, then 10ml 10% urea solution and 20 mL citrate buffer solution (pH 6.7) was added
131
After the incubation for 24 hours, the 38℃ of hot water was added to 100 mL scale line. The
132
the 3 mL of filtered soil solution was mixed with distillated water, sodium phenoxide, an
133
sodium hypochlorite solution. After 20 minutes, the mixed soil solution was diluted to 50 m
134
and determined at 578 nm. 135
Hydrolysable unidentified N = total hydrolysable N - amino acid N - ammonium N - amino
136
sugar N
(1)
137
Acid insoluble N = soil total N - total hydrolysable N
(2)
138
Statistical analysis
139
All data were related as means plus one standard deviation. One-way analysis of variance wa
140
used to examine the difference between treatments, and the significant difference was considere
141
to reach the p<0.05 level. Pearson correlation analysis was used for showing the relationshi
142
between soil organic N distribution and N-mineralizing enzyme activities. All statistical analys
143
was carried out by SPSS 19.0. 144
Results
145
Soil properties
146
As shown in Table 2. The contents of Cmic, Nmic, SOC, Ctot and Ntot increased gradually with th
147
increase in continuous cropping duration basically All of these indexes peaked at 20 years Th 127
2ml of the supernatant and ninhydrin solution mixed in the water bath kettle at 100℃ for 10
128
minutes and finally determined at 500 nm. For the measurement of urease activity, 5g of soil
129
samples and 1ml methylbenzene were added into 100 mL volumetric flask and incubated for 15
130
minutes, then 10ml 10% urea solution and 20 mL citrate buffer solution (pH 6.7) was added. 131
After the incubation for 24 hours, the 38℃ of hot water was added to 100 mL scale line. Then
132
the 3 mL of filtered soil solution was mixed with distillated water, sodium phenoxide, and
133
sodium hypochlorite solution. After 20 minutes, the mixed soil solution was diluted to 50 mL
134
and determined at 578 nm. Manuscript to be reviewed 135
Hydrolysable unidentified N = total hydrolysable N - amino acid N - ammonium N - amino
136
sugar N
(1) (1) (2) 139
All data were related as means plus one standard deviation. One-way analysis of variance was
140
used to examine the difference between treatments, and the significant difference was considered
141
to reach the p<0.05 level. Pearson correlation analysis was used for showing the relationship
142
between soil organic N distribution and N-mineralizing enzyme activities. All statistical analysis
143
was carried out by SPSS 19.0. 146
As shown in Table 2. The contents of Cmic, Nmic, SOC, Ctot and Ntot increased gradually with the
147
increase in continuous cropping duration basically. All of these indexes peaked at 20 years. The 146
As shown in Table 2. The contents of Cmic, Nmic, SOC, Ctot and Ntot increased gradually with the
147
increase in continuous cropping duration basically. All of these indexes peaked at 20 years. The Manuscript to be reviewed 148
Cmic of 20a increased 80.7%, 18.4%, and 36.4%, respectively, in comparison to 5a, 10a, 15a;
149
Nmic increased 92.1%, 20.9%, and 37.2%, respectively; Ctot increased 22.2%, 17.5%, and 6.6%,
150
respectively; Ntot increased 36.1%, 15.6%, and 8.4%, respectively; and SOC increased 44.7%,
151
13.6%, and 16.5%, respectively; all of which were significantly increased (P <0.05). However,
152
(C:N)mic and (C:N)tot tended to decrease with the increasing duration of continuous cropping, but
153
no significant difference was observed among treatments. The contents of Cmic, Nmic, Ctot, SOC,
154
and (C:N)tot decreased with the increase in continuous cropping duration, while the contents of
155
Ntot and (C:N)mic increased. 148
Cmic of 20a increased 80.7%, 18.4%, and 36.4%, respectively, in comparison to 5a, 10a, 15a;
149
Nmic increased 92.1%, 20.9%, and 37.2%, respectively; Ctot increased 22.2%, 17.5%, and 6.6%,
150
respectively; Ntot increased 36.1%, 15.6%, and 8.4%, respectively; and SOC increased 44.7%,
151
13.6%, and 16.5%, respectively; all of which were significantly increased (P <0.05). However,
152
(C:N)mic and (C:N)tot tended to decrease with the increasing duration of continuous cropping, but
153
no significant difference was observed among treatments. The contents of Cmic, Nmic, Ctot, SOC,
154
and (C:N)tot decreased with the increase in continuous cropping duration, while the contents of
155
Ntot and (C:N)mic increased. 156
Compared with the treatments without cotton residues incorporation, the content of Cmic,
157
Nmic, SOC, Ctot and Ntot in the treatments with cotton residues incorporation was significantly
158
higher. Compared with CK5, CK10, and CK20, Cmic increased 4.2%, 65.3%, and 125.1%, Nmic
159
increased 3.8%, 73.6%, and 146.7%, SOC increased 10.6%, 49.5%, and 95.9% in 5a, 10a, and
160
20a, respectively, and with the exception of 5a, these values were all significant (P <0.05). 161
Additionally, the content of Ctot increased 17.4%, 24.1%, and 62.8%, and that of Ntot increased
162
26.7%, 39.4%, and 44.4%, respectively (P <0.05). Cotton residues incorporation thus had no
163
significant effect on (C:N)mic and (C:N)tot. 164
Soil organic N fractions 165
The contents of different fractions of soil organic N are shown in Table 3. Under the condition
166
of cotton residues incorporation, amino acid N and amino sugar N, ammonium N, hydrolysable
167
unidentified N, and acid-insoluble N increased gradually with increased continuous cropping
168
basically. All of these indexes reached their maximum value after 20 years of continuous PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed 169
cropping. In 5a, 10a, and 15a, the amino acid N content at 20 years of continuous cropping
170
increased 52.9%, 13.0%, and 23.8%, respectively; ammonium N increased 34.5%, 16.4%, and
171
8.3%, respectively; amino sugar N increased 42.2%, 11.3%, and 21.9%, respectively;
172
hydrolysable unidentified N and acid-insoluble N increased 32.1%, 15.1%, -3.8%, and 28.9%,
173
18.4%, and 8.7%, respectively. The difference between treatments were significant (P < 0.05). In
174
the absence of cotton residues incorporation, amino acid N and ammonium N. Hydrolysable
175
unidentified N and acid-insoluble N also increased with the increase in continuous cropping
176
duration, but amino sugar N decreased gradually, the difference was not significant. Amino acid
177
N, ammonium N, amino sugar N, hydrolysable unidentified N, and acid-insoluble N in the
178
treatment with cotton residues incorporation were significantly higher than that of the treatment
179
without cotton residues incorporation. Compared with CK5, CK10, and CK20, the amino acid N
180
of 5a, 10a, and 20a increased 26.87%, 53.33%, and 48.57%; ammonium N increased 8.7%, 7.7%,
181
and 28.6%; amino sugar N increased 11.1%, 53.3%, and 96.9%; hydrolysable unidentified N
182
increased 32.4%, 43.9%, and 48.3%; and acid-insoluble N increased 29.5%, 34.5%, and 38.8%,
183
respectively. 184
The ratio of amino acid N to total N was 19.5%-22.4%; ammonium N was 16.3%-18.7%;
185
amino sugar N was 4.0%-5.9% (Fig. 1); hydrolysable unidentified N was 25.7%-29.8%; and
186
acid-insoluble N was 29.2%-31.5%, respectively. The organic N fractions were in the following
187
order: acid-insoluble N > hydrolysable unidentified N > amino acid N > ammonium N > amino
188
sugar N. The proportion of amino acid N, acid sugar N, and hydrolysable acid N under cotton
189
residues incorporation increased compared with the treatment lacking cotton residues. Compared Manuscript to be reviewed 217
Discussion 215
proportion of amino acid N, amino sugar N, and hydrolyzed unidentified N were
216
correlated with all the N-mineralizing enzymes, while acid-insoluble N was negatively c
217
Discussion
218
Effect of cotton residues incorporation on soil properties and organic N fractions
219
The previous study showed that the content of soil total C, N, and SOC in the cotton
220
incorporation treatments increased with the duration of continuous cotton cropping
221
and our finding are consistent with Yu et al. (2020). On the contrary, in the treatment
222
cotton residues were not incorporated into field, the content of soil total C and SOC
223
with the increasing duration of continuous cotton cropping. This is mainly due to
224
organic C content in the cotton residues, which provides exogenous organic matter to th
225
partly enters the soil during microbial decomposition, thus increasing the soil organic Manuscript to be reviewed 190
with CK5, CK10, and CK20, the proportion of amino acid N increased 0.1%, 10.0%, and 2.9%;
191
acid sugar N increased -12.3%, 10.0% and 36.3%; and the proportion of hydrolysable
192
unidentified N increased 4.5%, 3.3%, and 2.7%, respectively, under continuous cropping for 5,
193
10, and 20 years. In contrast, the proportion of ammonium N and acid-insoluble N decreased
194
6.6%, 12.6%, and 8.5% and -2.3%, 3.45%, and 3.9%, respectively, for 5, 10, and 20 years of
195
continuous cropping, compared with CK5, CK10, and CK20. The proportion of amino acid N
196
increased, but the proportion of acid-insoluble N decreased with the increase in continuous
197
cropping duration. 198
Soil N-mineralizing enzyme activities 199
As shown in Fig. 2 and Fig. 3, the protease and urease showed an increasing trend with the
200
increase in cropping duration under the cotton residues incorporation treatment, with values of
201
18.6 µg g-1 and 550.3 µg g-1, respectively, for 20 years of continuous cropping. Compared with
202
continuous cropping for 5a, 10a, and 15a, the protease activities of continuous cropping for 20
203
years increased significantly (P < 0.05) 34.2%, 22.5%, and 26.3%, respectively. Urease contents
204
under continuous cropping increased 15.5%, 2.7%, and 1.6%, respectively, and the 5-year
205
treatment showed a significant difference. The activities of the two enzymes all showed a
206
decreasing trend with the increase in continuous cropping duration in the absence of cotton
207
residues incorporation. The enzyme activities under cotton residues incorporation were
208
significantly higher in comparison for the same continuous cropping treatment durations. 209
Compared with CK5, CK10, and CK20, protease activity under continuous cropping for 5a, 10a,
210
and 20a increased 11.8%, 30.4%, and 53.4 %, respectively; urease increased 10.3%, 37.6%, and PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed 211
53.1%, respectively. 211
53.1%, respectively. 212
Relationship between soil organic N fractions and N-mineralizing enzyme activities
213
The total organic N and proportion of ammonium N showed significant positive and
214
correlations with all N-mineralizing enzymes (protease, urease) (P < 0.05) (Table
215
proportion of amino acid N, amino sugar N, and hydrolyzed unidentified N were p
216
correlated with all the N-mineralizing enzymes, while acid-insoluble N was negatively co
217
Discussion
218
Effect of cotton residues incorporation on soil properties and organic N fractions
219
The previous study showed that the content of soil total C, N, and SOC in the cotton
220
incorporation treatments increased with the duration of continuous cotton cropping in
221
and our finding are consistent with Yu et al. (2020). On the contrary, in the treatments
222
cotton residues were not incorporated into field, the content of soil total C and SOC d
223
with the increasing duration of continuous cotton cropping. This is mainly due to
224
organic C content in the cotton residues, which provides exogenous organic matter to the
225
partly enters the soil during microbial decomposition, thus increasing the soil organic C
226
(Xu et al., 2011). Secondly, because of the high organic C content of the cotton residues
227
high C:N ratio, microorganisms need to absorb more inorganic N from the soil to sati
228
growth requirements, thus improving the ability of microorganisms to utilize ammonium
229
nitrate N (Ren et al., 2016). Microorganisms are required for the assimilation of more a
230
N into the soil organic N pool (Tian et al., 2017). Cotton residues incorporation incre
231
content of C and N in the soil, which indicated the N in the cotton residues incor 212
Relationship between soil organic N fractions and N-mineralizing enzyme activities 212
Relationship between soil organic N fractions and N-mineralizing enzyme activities 213
The total organic N and proportion of ammonium N showed significant positive and negative
214
correlations with all N-mineralizing enzymes (protease, urease) (P < 0.05) (Table 4). The
215
proportion of amino acid N, amino sugar N, and hydrolyzed unidentified N were positively
216
correlated with all the N-mineralizing enzymes, while acid-insoluble N was negatively correlated. 218
Effect of cotton residues incorporation on soil properties and organic N fractions 218
Effect of cotton residues incorporation on soil properties and organic N fractions 219
The previous study showed that the content of soil total C, N, and SOC in the cotton residues
220
incorporation treatments increased with the duration of continuous cotton cropping increased,
221
and our finding are consistent with Yu et al. (2020). On the contrary, in the treatments that the
222
cotton residues were not incorporated into field, the content of soil total C and SOC decreased
223
with the increasing duration of continuous cotton cropping. This is mainly due to the high
224
organic C content in the cotton residues, which provides exogenous organic matter to the soil and
225
partly enters the soil during microbial decomposition, thus increasing the soil organic C content
226
(Xu et al., 2011). Secondly, because of the high organic C content of the cotton residues, under a
227
high C:N ratio, microorganisms need to absorb more inorganic N from the soil to satisfy their
228
growth requirements, thus improving the ability of microorganisms to utilize ammonium N and
229
nitrate N (Ren et al., 2016). Microorganisms are required for the assimilation of more available
230
N into the soil organic N pool (Tian et al., 2017). Cotton residues incorporation increased the
231
content of C and N in the soil, which indicated the N in the cotton residues incorporation Manuscript to be reviewed 253
stable N compounds. Stable organic N is difficult to be mineralized, leading to its accumulation
254
in the soil (Tian et al., 2017). Secondly, acid-insoluble N is considered as the structural
255
component of humus, and its main source is the senescent substances in aboveground and
256
underground debris (Ren et al., 2016). This can be explained by the increase in humus content in
257
the soil following cotton residues incorporation, leading to the increase in acid-insoluble N
258
content. Manuscript to be reviewed 232
treatment were significantly higher than that lacking cotton residues, which may be due to the
233
increase in soil microbial biomass caused by cotton residues incorporation and the improved soil
234
microbial structure. As the structural index of soil microbial community, the (C:N)mic ratio is
235
used to describe the relative contribution of bacteria and fungi to the soil microbial community, a
236
higher (C:N)mic ratio indicates that the proportion of fungi in the soil microbial community is
237
higher. The lower (C:N)mic ratio indicates that the proportion of bacteria is higher (Tian et al.,
238
2017). In this study, the (C:N)mic ratio decreased gradually with the increased in continuous
239
cropping duration under cotton residues incorporation treatments, while the opposite was
240
observed in the treatments that the cotton residues were not incorporated. The results showed
241
that the number of bacteria in the soil increased gradually with the increase in continuous
242
cropping duration under cotton residues incorporation, which will be beneficial to the
243
development of the cotton field soil in a good direction. 244
This study also found that cotton residues incorporation increased the content of acid-
245
hydrolyzed ammonium N, and the content increased gradually with the increase in continuous
246
cropping duration. As an important component of soil organic N decomposition, ammonium N
247
also partly originates from soil-fixed NH4+, which constitutes a rapidly released and available
248
soil N pool for plants and microorganisms (Lü et al., 2013). The decrease in the proportion of
249
ammonium N to total N under cotton residues incorporation could be attributed to the increased
250
fixation or uptake of mineral N by microorganisms and plants. The acid-insoluble N measured
251
by Bremner’s method is in the form of heterocyclic N or a combination of a heterocyclic
252
compound with an aromatic compound. Heterocyclic compounds and aromatic compounds are 259
Effect of cotton residues incorporation on potential N-mineralizing enzyme activities 260
Enzymes mainly originate from soil microorganisms and plant secretions in the soil and are the
261
main catalyst of all biochemical processes in the soil. Enzyme activity not only reflects soil
262
quality, but also reflects the direction and intensity of biochemical reactions in the soil (He et al.,
263
2020). It is considered as a sensitive index of soil system change because of the responsiveness,
264
rapid determination, specificity, and comprehensiveness of soil enzymes. Our study showed that
265
cotton residues incorporation could increase the soil urease and protease activities, which was
266
due to the increase in soil urease and protease activities, which was due to the increase in soil
267
microbial C under cotton residues incorporation treatments. On the contrary, the soil microbial C
268
decreased gradually with the increasing duration of continuous cropping when the cotton
269
residues were not incorporated, leading to the decreased soil enzyme activity. 260
Enzymes mainly originate from soil microorganisms and plant secretions in the soil and are the
261
main catalyst of all biochemical processes in the soil. Enzyme activity not only reflects soil
262
quality, but also reflects the direction and intensity of biochemical reactions in the soil (He et al.,
263
2020). It is considered as a sensitive index of soil system change because of the responsiveness,
264
rapid determination, specificity, and comprehensiveness of soil enzymes. Our study showed that
265
cotton residues incorporation could increase the soil urease and protease activities, which was
266
due to the increase in soil urease and protease activities, which was due to the increase in soil
267
microbial C under cotton residues incorporation treatments. On the contrary, the soil microbial C
268
decreased gradually with the increasing duration of continuous cropping when the cotton
269
residues were not incorporated, leading to the decreased soil enzyme activity. 272
The transformation and absorption of soil N is conducted by enzyme systems, and their synthesis
273
and expression require C, N, and energy, suggesting that the form of available N, the source of C, Manuscript to be reviewed 274
and the C availability in relation to N are important factors (Yang et al., 2016). It is generally
275
believed that all organic N is mineralized to NH4+ before being absorbed by soil microorganisms. 276
This pathway is generally known as the mineralization-immobilization-turnover (MIT) route. 277
When the concentration of NH4+ was relatively high (Figure S1), the gene transcription of the
278
enzyme systems needed to replace the N source were inhibited, and cell N-mineralizing enzymes
279
were involved in the potential transformation of soil organic N under the condition of cotton
280
residues incorporation, enzymatic mineralization is superior to the direct route (Tian et al., 2017). 281
Under C-limited conditions, the substrates of N with low molecular weights, such as amino acids
282
and amino sugars, can be directly absorbed by microorganisms as a C source (Nannipieri &
283
Eldor, 2009). There were significant positive correlations between soil total organic N and N-
284
mineralizing enzyme activities, indicating that the activities of N-mineralizing enzymes in the
285
soil played an active role in the potential transformation of soil organic N. However, there was
286
no significant positive correlation between the ratios of amino acid N, amino sugar N, and acid-
287
hydrolyzed unknown N and the activity of N-mineralizing enzymes, which was due to the fact
288
that the transformation of soil organic N was restricted by the lack of C source. But the ratio of
289
ammonia N showed a significant negative correlation with the activity of N-mineralizing
290
enzymes. This is because the increased soil N-mineralizing enzymes after cotton residues
291
incorporation promoted the mineralization of soil organic N. 292
In the cotton residues incorporation treatments, the decrease of the proportion was attributed
293
to the increase of fixation or absorption of mineral N by microorganisms and plants. Therefore, 292
In the cotton residues incorporation treatments, the decrease of the proportion was attributed
293
to the increase of fixation or absorption of mineral N by microorganisms and plants. Therefore,
294
in the soil of long-term continuous cropping cotton field, soil microorganisms need to absorb Manuscript to be reviewed 295
organic N molecules to meet the demand for C due to cotton residues incorporation. 296
Conclusions
297
Cotton residues incorporation was beneficial to improve soil quality and soil fertility in the long-
298
term continuous cotton cropping field. The benefits can be increased over time. Under the
299
condition of cotton residues incorporation, when the concentration of NH4+ was high, enzymatic
300
mineralization constituted the main pathway of potential organic N turnover. However, when
301
cotton residues were not incorporated into the soil, the utilization of soil organic N was the most
302
direct route due to the lower soil organic C availability. 295
organic N molecules to meet the demand for C due to cotton residues incorporation. 296
Conclusions
297
Cotton residues incorporation was beneficial to improve soil quality and soil fertility in the long-
298
term continuous cotton cropping field. The benefits can be increased over time. Under the
299
condition of cotton residues incorporation, when the concentration of NH4+ was high, enzymatic
300
mineralization constituted the main pathway of potential organic N turnover. However, when
301
cotton residues were not incorporated into the soil, the utilization of soil organic N was the most
302
direct route due to the lower soil organic C availability. 303
304
Acknowledgements
305
This study is supported by the National Natural Science Foundation of China (31960396,
306
31560375). 307
308
References
309
Ashton IW, Miller AE, Bowman WD, Suding KN. 2010. Niche complementarity due to
310
plasticity in resource use: plant partitioning of chemical N forms. Ecology 91: 3252-3260. 311
Bakht J, Shafi M, Jan MT. 2009. Influence of crop residue management, cropping system and
312
N fertilizer on soil N and C dynamics and sustainable wheat production. Soil & Tillage
313
Research 104: 233-240. 314
Bremner JM. 1965. Organic forms of nitrogen//Black C A, ed. Methods of soil analysis. 315
Madison: American Society of Agronomy, pp: 1238-1255. PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) 297
Cotton residues incorporation was beneficial to improve soil quality and soil fertility in the long-
298
term continuous cotton cropping field. The benefits can be increased over time. Under the
299
condition of cotton residues incorporation, when the concentration of NH4+ was high, enzymatic
300
mineralization constituted the main pathway of potential organic N turnover. Manuscript to be reviewed However, when
301
cotton residues were not incorporated into the soil, the utilization of soil organic N was the most
302
direct route due to the lower soil organic C availability. 309
Ashton IW, Miller AE, Bowman WD, Suding KN. 2010. Niche complementarity due to
310
plasticity in resource use: plant partitioning of chemical N forms. Ecology 91: 3252-3260. 314
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Research, 196: 104483. PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Table 1(on next page) Basic soil properties at experimental site. PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Table 2(on next page) Table 2(on next page) Manuscript to be reviewed 1 1
Duration
Bulk
density
pH
Organic matter
Alkali-hydrolyzable
nitrogen
Available
phosphorus
Available potassium
(a)
(g cm-3)
(g·kg-1)
(mg·kg-1)
(mg·kg-1)
(mg·kg-1)
5
1.37
7.31
15.28
70.84
40.8
183
10
1.38
7.24
16.32
74.36
23.1
110.3
15
1.35
7.35
17.98
84.06
25.4
65.5
20
1.33
7.26
18.51
89.08
20.1
72.3
CK5
1.31
7.29
14.35
113.1
36.47
125.4
CK10
1.36
7.33
15.67
98.75
46.28
112.8
CK20
1.34
7.37
15.22
109.4
40.34
108.5
2 PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed 1
Treatment
Cmic
Nmic
Ctot
Ntot
SOC
(C:N)mic
(C:N)tot
mg kg-1
g kg-1
5 a
83.88 ± 5.21d
18.5 ± 0.83d
10.25 ± 0.79c
0.51 ±0.02d
3.44 ± 0.51c
4.57 ± 0.36ab
20.21 ± 3.21a
10 a
127.54 ± 8.35b
29.41 ± 2.61b
10.66 ± 3.51c
0.60 ± 0.02c
4.38 ± 0.67b
4.36 ± 0.78ab
17.87 ± 3.71b
15 a
110.75 ± 8.26c
25.9 ± 4.48c
11.75 ± 3.67b
0.64 ± 0.03b
4.27 ± 1.02b
4.29 ± 1.15ab
18.48 ± 2.57a
20 a
151.04 ± 10.38a
35.55 ± 6.50a
12.52 ± 2.16a
0.69 ± 0.02a
4.98 ± 0.77a
4.19 ± 0.22b
18.19 ± 5.10ab
CK5
80.52 ± 3.67d
17.82 ± 3.33d
8.73 ± 1.61d
0.40 ± 0.04g
3.11 ± 0.58cd 4.54 ± 0.73ab
21.7 ± 4.18a
CK10
77.16 ± 4.88de
16.94 ± 6.20de 8.59 ± 2.30de
0.43 ± 0.02f
2.93 ± 0.60d
4.58 ± 0.80ab
19.96 ± 1.15a
CK20
67.09 ± 5.12e
14.41 ± 3.52e
7.69 ± 2.23e
0.48 ± 0.04e
2.54 ± 0.39e
4.71 ± 0.54a
16.18 ± 1.06b
2
Sample size n=3. Value are means ± standard errors. Upper and lower case indicated significant differences (P < 0.05) between treatments. Table 2(on next page) Changes of soil properties in 0-40 cm soil depth after long-term continuous croppingand
cotton residues incorporation. PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed Manuscript to be reviewed 3
5 a: Cotton residues have been incorporated for 5 years in long-term continuous cropping cotton field; 10 a: Cotton residues have been incorporated
4
for 5 years in long-term continuous cropping cotton field; 15 a: Cotton residues have been incorporated for 15 years in long-term continuous
5
cropping cotton field; 20 a: Cotton residues have been incorporated for 20 years in long-term continuous cropping cotton field。
6 1
Treatment
Cmic
Nmic
Ctot
Ntot
SOC
(C:N)mic
(C:N)tot
mg kg-1
g kg-1
5 a
83.88 ± 5.21d
18.5 ± 0.83d
10.25 ± 0.79c
0.51 ±0.02d
3.44 ± 0.51c
4.57 ± 0.36ab
20.21 ± 3.21a
10 a
127.54 ± 8.35b
29.41 ± 2.61b
10.66 ± 3.51c
0.60 ± 0.02c
4.38 ± 0.67b
4.36 ± 0.78ab
17.87 ± 3.71b
15 a
110.75 ± 8.26c
25.9 ± 4.48c
11.75 ± 3.67b
0.64 ± 0.03b
4.27 ± 1.02b
4.29 ± 1.15ab
18.48 ± 2.57a
20 a
151.04 ± 10.38a
35.55 ± 6.50a
12.52 ± 2.16a
0.69 ± 0.02a
4.98 ± 0.77a
4.19 ± 0.22b
18.19 ± 5.10ab
CK5
80.52 ± 3.67d
17.82 ± 3.33d
8.73 ± 1.61d
0.40 ± 0.04g
3.11 ± 0.58cd 4.54 ± 0.73ab
21.7 ± 4.18a
CK10
77.16 ± 4.88de
16.94 ± 6.20de 8.59 ± 2.30de
0.43 ± 0.02f
2.93 ± 0.60d
4.58 ± 0.80ab
19.96 ± 1.15a
CK20
67.09 ± 5.12e
14.41 ± 3.52e
7.69 ± 2.23e
0.48 ± 0.04e
2.54 ± 0.39e
4.71 ± 0.54a
16.18 ± 1.06b
2
Sample size n=3. Value are means ± standard errors. Upper and lower case indicated significant differences (P < 0.05) between treatments. Manuscript to be reviewed Table 3(on next page) Manuscript to be reviewed 3
5 a: Cotton residues have been incorporated for 5 years in long-term continuous cropping cotton field; 10 a: Cotton residues have been incorporated
4
for 5 years in long-term continuous cropping cotton field; 15 a: Cotton residues have been incorporated for 15 years in long-term continuous
5
cropping cotton field; 20 a: Cotton residues have been incorporated for 20 years in long-term continuous cropping cotton field。
6 3
5 a: Cotton residues have been incorporated for 5 years in long-term continuous cropping cotton field; 10 a: Cotton residues have been incorporated
4
for 5 years in long-term continuous cropping cotton field; 15 a: Cotton residues have been incorporated for 15 years in long-term continuous
5
cropping cotton field; 20 a: Cotton residues have been incorporated for 20 years in long-term continuous cropping cotton field。 3
5 a: Cotton residues have been incorporated for 5 years in long-term continuous cropping cotton field; 10 a: Cotton residues have been incorporated
4
for 5 years in long-term continuous cropping cotton field; 15 a: Cotton residues have been incorporated for 15 years in long-term continuous
5
cropping cotton field; 20 a: Cotton residues have been incorporated for 20 years in long-term continuous cropping cotton field。 PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Table 3(on next page) Table 3(on next page) Concentrations of N (mg kg-1) in the various fractions in long-term continuous
croppingcotton field under straw incorporation PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed Manuscript to be reviewed Table 4(on next page) Table 4(on next page) Manuscript to be reviewed Treatment
Hydrolysable N
Insoluble N
Amino acid N
Ammonium N
Amino sugar N
Unidentified N
Total N
5 a
99.17 ± 10.11c
84.58 ± 6.90c
26.25 ± 3.86cd
138.25 ± 14.17c
348.25 ± 5.25c
160.32 ± 10.22c
10 a
134.17 ± 5.05b
97.71 ± 2.53c
33.54 ± 5.05ab
158.67 ± 12.75b
424.08 ± 17.56b
174.49 ± 10.31b
15 a
122.5 ± 10.11bc
105.00 ± 7.58b
30.63 ± 7.58bc
189.88 ± 6.97a
448.00 ± 16.33b
190.10 ± 12.28ab
20 a
151.67 ± 10.11a
113.75 ± 4.38a
37.33 ± 3.54a
182.58 ± 15.67a
485.33 ± 12.63a
206.57 ± 12.08a
CK5
78.17 ± 10.31e
71.46 ± 5.05d
23.63 ± 2.87de
104.42 ± 19.69d
277.67 ± 10.34e
123.70 6 ± 14.99d
CK10
87.50 ± 5.52cd
80.21 ± 5.05cd
21.88 ± 4.35de
110.25 ± 10.67d
299.83 ± 9.02de
129.69 ± 10.16d
CK20
102.08 ± 12.39c
86.04 ± 3.65c
18.96 ± 2.53e
123.08 ± 16.88cd
330.17 ± 9.30cd
148.88 ± 5.11cd
1
Sample size n=3. Value are means ± standard errors. Upper and lower case indicated significant differences (P < 0.05) between treatments. 2 PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed Table 4(on next page) Correlations between soil organic N distribution (%) and N-mineralizing enzymeactivities
in the long-term continuous cropping cotton field. PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed Manuscript to be reviewed Protease
Urease
Amino acid N (%)
0.293
0.157
Ammonium N (%)
-0.784*
-0.909**
Amino sugar N (%)
0.513
0.47
Unidentified N (%)
0.346
0.608
Insoluble N (%)
-0.546
-0.612
Total organic N (%)
0.837*
0.854*
1
* and **: statistically significant at P < 0.05 and 0.01, respectively;
2 PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Manuscript to be reviewed Manuscript to be reviewed Figure 1 Distribution (%) of soil organic N fractions in long-term continuous cropping cotton field
under straw incorporation. AAN, AN, ASN, HUN, AIN AAN, AN, ASN, HUN, AIN PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Figure 2 Changes of soil protease activities in long-term continuous cropping cotton field under
straw incorporation. Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021) Figure 3 Changes of soil urease activities in long-term continuous cropping cotton field under
straw incorporation. PeerJ reviewing PDF | (2020:06:50331:2:0:NEW 29 Jan 2021)
|
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